Methods and compositions for the ex vivo replication of human hematopoietic stem cells

ABSTRACT

Methods, including culture media conditions, which provide for ex vivo human stem cell division and stable genetic transformation and/or the optimization of human hematopoietic progenitor cell cultures and/or increasing the metabolism or GM-CSF secretion or IL-6 secretion of human stromal cells are disclosed. The methods rely on culturing human stem cells and/or human hematopoietic progenitor cells and/or human stromal cells in a liquid culture medium which is replaced, preferably perfused, either continuously or periodically, at a rate of 1 ml of medium per ml of culture per about 24 to about 48 hour period, and removing metabolic products and replenishing depleted nutrients while maintaining the culture under physiologically acceptable conditions. Optionally growth factors are added to the culture medium.

This is a Continuation of application Ser. No. 07/740,590 filed on Aug.5, 1991, now U.S. Pat. No. 5,399,493, which was a continuation in partof application Ser. No. 07/628,343, filed on Dec. 17, 1990, nowabandoned, which was a continuation-in-part of application Ser. No.07/366,639, filed on Jun. 15, 1989, now abandoned, which was filed asInternational Application No. PCT/US90/03438, on Jun. 14/1990, and is aCIP of Ser. No. 07/737,024 filed Jul. 29, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods and compositions for the growth andtransformation of mammalian cells in culture, particularly the growthand transformation of hematopoietic cell cultures.

2. Discussion of the Background

All of the circulating blood cells in the normal adult, includingerythrocytes, leukocytes, platelets and lymphocytes, originate asprecursor cells within the bone marrow. These cells, in turn, derivefrom very immature cells, called progenitors, which are assayed by theirdevelopment into contiguous colonies of mature blood cells in 1-3 weekcultures in semisolid media such as methylcellulose or agar. Progenitorcells themselves derive from a class of progenitor cells called stemcells. Stem cells have the capacity, upon division, for bothself-renewal and differentiation into progenitors. Thus, dividing stemcells generate both additional primitive stem cells and somewhat moredifferentiated progenitor cells. In addition to the generation of bloodcells, stem cells also may give rise to osteoblasts and osteoclasts, andperhaps cells of other tissues as well. This document described methodsand compositions which permit, for the first time, the successful invitro culture of human hematopoietic stem cells, which results in theirproliferation and differentiation into progenitor cells and more matureblood cells.

In the late 1970s the liquid culture system was developed for growinghematopoietic bone marrow in vitro. The cultures are of great potentialvalue both for the analysis of normal and leukemic hematopoiesis and forthe experimental manipulation of bone marrow, for, e.g.,retroviral-mediated gene transfer. These cultures have allowed adetailed analysis of murine hematopoiesis and have resulted in adetailed understanding of the murine system. In addition, it has madepossible retroviral gene transfer into cultured mouse bone marrow cells.This allowed tagging murine hematopoietic cells proving the existence ofthe multi-potent stem cell and of the study of the various genes in theprocess of leukemogenesis.

But while it has been possible to transfer retroviral genes intocultured mouse bone marrow cells, this is not yet been possible incultured human bone marrow cells because, to date, human long-term bonemarrow cultures have been limited both in their longevity and in theirability to maintain stem cell survival and their ability to produceprogenitor cells over time.

Human liquid bone marrow cultures were initially found to have a limitedhematopoietic potential, producing decreasing numbers of progenitorcells and mature blood cells, with cell production ceasing by 6 to 8weeks. Subsequent modifications of the original system resulted only inmodest improvements. A solution to this problem is of incalculable valuein that it would permit, e.g., expanding human stem cells and progenitorcells for bone marrow transplantation and for protection fromchemotherapy, selecting and manipulating such cells, i.e., for genetransfer, and producing mature human blood cells for transfusiontherapy.

Studies of hematopoiesis and in vitro liquid marrow cultures haveidentified fibroblasts and endothelial cells within adhering layers ascentral cellular stromal elements. These cells both provide sites ofattachment for developing hematopoietic cells and can be induced tosecrete hematopoietic growth factors which stimulate progenitor cellproliferation and differentiation. These hematopoietic growth factorsinclude granulocyte colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF) andinterleukin-6 (IL-6).

Cultures of human bone marrow cells on such adherent layers in vitrohowever have been largely disappointing. Unlike related cultures fromother species, such as mouse and tree shrew, human liquid marrowcultures fail to produce significant numbers of either nonadherenthematopoietic precursor cells or clonogenic progenitor cells for over 6to 8 weeks. And although cultures lasting 3-5 months have been reported,no culture which stably produces progenitor cells from stem cellscontinuously for more than 4-6 weeks has been reported.

Moreover, nonadherent and progenitor cell production typically declinedthroughout even the short life of these cultures, so that it is notclear that stem cell survival or proliferation is supported at all bythese cultures. Further, when studied in isolation, unstimulated bonemarrow stromal cells secrete little if any detectable hematopoieticgrowth factors (HGFs).

The lack of stable progenitor Cell and mature blood cell production inthese cultures has led to the belief that they are unable to supportcontinual stem cell renewal and expansion. It has therefore beenpresumed that the cultures either lack a critical stem cell stimulant(s)and/or contain a novel stem cell inhibitor(s). But while explanationsfor failure to detect HGFs and uninduced stromal cell cultures have beensuggested, the null hypothesis, which combines the failure to detectHGFs and the relative failure of human liquid marrow cultures, would bethat the culture systems used in vitro do not provide the full range ofhematopoietic supportive function of adherent bone marrow stromal cellsin vivo.

Stem cell and progenitor cell expansion for bone marrow transplantationis a potential application for human long-term bone marrow cultures.Human autologous and allogenic bone marrow transplantation are currentlyused as therapies for diseases such as leukemia, lymphoma and otherlife-threatening disorders. For these procedures however, a large amountof donor bone marrow must be removed to insure that there is enoughcells for engraftment.

A culture providing stem cell and progenitor cell expansion would reducethe need for large bone marrow donation and would make possibleobtaining a small marrow donation and then expanding the number of stemcells and progenitor cells in vitro before infusion into the recipient.Also, it is known that a small number of stem cells and progenitor cellscirculate in the blood stream. If these stem cells and progenitor cellscould be collected by phoresis and expanded, then it would be possibleto obtain the required number of stem cells and progenitor cells fortransplantation from peripheral blood and eliminate the need for bonemarrow donation.

Bone marrow transplantation requires that approximately 1×10⁸ to 2×10⁸bone marrow mononuclear cells per kilogram of patient weight be infusedfor engraftment. This requires the bone marrow donation of the samenumber of cells which is on the order of 70 ml of marrow for a 70 kgdonor. While 70 ml is a small fraction of the donors marrow, it requiresan intensive donation and significant loss of blood in the donationprocess. If stem cells and progenitor cells could be expanded ten-fold,the donation procedure would be greatly reduced and possibly involveonly collection of stem cells and progenitor cells from peripheral bloodand expansion of these stem cells and progenitor cells.

Progenitor cell expansion would also be useful as a supplementaltreatment to chemotherapy, and is another application for humanlong-term bone marrow cultures. The dilemma faced by oncologist is thatmost chemotherapy agents used to destroy cancer act by killing all cellsgoing through cell division. Bone marrow is one of the most prolifictissues in the body and is therefore often the organ that is initiallydamaged by chemotherapy drugs. The result is that blood cell productionis rapidly destroyed during chemotherapy treatment and chemotherapy mustbe terminated to allow the hematopoietic system to replenish the bloodcell supply before a patient is retreated with chemotherapy. It may takea month or more for the once quiescent stem cells to raise up the whiteblood cell count to acceptable levels to resume chemotherapy duringwhich case the drop in blood cell count is repeated. Unfortunately,while blood cells are regenerating between chemotherapy treatments, thecancer has time to grow and possibly become more resistant to thechemotherapy drugs due to natural selection.

To shorten the time between chemotherapy treatments, large numbers ofprogenitor and immature blood cells could be given back to the patient.This would have the effect of greatly reducing the time the patientwould have low blood cell counts, thereby allowing more rapid resumptionof the chemotherapy treatment. The longer chemotherapy is given and theshorter the duration between treatments, the greater the odds ofsuccessfully killing the cancer.

The hematopoietic cells required for progenitor cell expansion may comefrom either bone marrow withdrawal or peripheral blood collection. Bonemarrow harvests would result in collection of approximately 4×10⁵ CFU-GMprogenitor cells. Phoresis of 5 liters of peripheral blood would collectapproximately 10⁵ CFU-GM although this number could be increased to 10⁶CFU-GM by prior treatment of the donor with GM-CSF. Rapid recovery of apatient would require transfusion of approximately 1×10⁸ to 5×10⁸ CFU-GMwhich is 100 to 1,000 times more than obtained by routine bone marrowdonation or by peripheral blood donation. Therefore, expansion of bonemarrow or peripheral blood to increase the number of CFU-GM 2 to 3orders of magnitude would significantly affect chemotherapyadministration and Cancer treatment.

Gene therapy is a rapidly growing field in medicine which is also ofinestimable clinical potential. Gene therapy has many potential uses intreating disease and has been reviewed extensively. See, e.g., Boggs,Int. J. Cell Cloning. (1990) 8:80-96, Kohn et al, Cancer Invest. (1989)7 (2):179-192, Lehn, Bone Marrow Transp. (1990) 5:287-293, and Verma,Scientific Amer. (1990) pp. 68-84. Genetically transformed human stemcells have wide potential application in clinical medicine, as agents ofgene therapy.

Gene therapy describes an emerging approach to clinical treatment whichhas evolved from earlier approaches in medical care. The earliestapproaches to medical care, evolving over centuries, included grosssurgical procedures and the administration of crude mixtures asmedicinal agents. In the past century, biochemical pharmacology hassupervened as the major method of medical treatment. Under thisparadigm, pure biochemical molecules are delivered to the patient. Ingeneral, such pharmacologic agents act either as poisons (such asantimicrobials or cancer chemotherapy agents), physiologic mimeticswhich stimulate endogenous receptors (e.g., opiates, adrenergicagonists), or physiologic antagonists which block endogenous receptors(e.g. antihypertensives, anaesthetics).

Gene therapy is, by definition, the insertion of genes into cells forthe purpose of medicinal therapy. The principle underlying gene therapyis to, rather than deliver doses of pharmacologic molecules, deliver afunctional gene whose RNA or protein product will produce the desiredbiochemical effect in the target cell or tissue. There are severalpotential advantages of gene therapy over classical biochemicalpharmacology. First, inserted genes can produce extremely complexmolecules, including RNA and protein, which can be extraordinarilydifficult or impossible to administer and deliver themselves. Next,controlled insertion of the desired gene into specific target cells cancontrol the production of gene product to defined tissues. Finally, genetherapy can in principle be permanent within an individual, as the genewill continue to function in the target cells and their progeny.

There are several problems that must therefore be addressed forsuccessful gene therapy. The first is to be able to insert the desiredtherapeutic gene into the chosen cells. Second, the gene must beadequately expressed in the target cell, resulting in the appropriatelevels of gene product. Finally the RNA or protein produced must beproperly processed by the target cell so that it is functional, i.e. sothat gene therapy actually infers clinical therapy. Several methods ofgene insertion into human cells in vitro are listed in Table 1.

                  TABLE 1                                                         ______________________________________                                        Comparison of DNA transfer methods.                                           Variable  Microinjection                                                                           Electroporation                                                                          Retrovirus                                    ______________________________________                                        Efficiency                                                                              10-100%    0.0001-1%  1-100%                                                                        (depends on titer)                            Effort    High       Low        Intermediate                                  Expense   High       Low        Intermediate                                  Stability Good       Good       May be inactivated                                                            or become infective                           DNA synthesis                                                                           ?          ?          Required                                      Size of DNA                                                                             Not restricted                                                                           Not restricted                                                                           Limited (≦8 kb)                        input                                                                         Need extraneous                                                                         No         No         Yes                                           DNA                                                                           ______________________________________                                    

Other techniques, such as homologous recombination, are being developedas well in many laboratories. Research in gene therapy has been on-goingfor several years in several types of cells in vitro, progressed toanimal studies, and has recently entered the first human clinicaltrial(s).

The hematopoietic system is an ideal choice as a delivery system forgene therapy. Hematopoietic cells are readily accessible, simply by bonemarrow aspiration or by peripheral blood mononuclear cell harvest. Oncethe genetic insertion is accomplished in vitro the treated cells can bereinfused intravenously, after which the genetically transformed cellswill home to and develop in the bone marrow. Since mature blood cellscirculate throughout the body, the genetically modified cells candeliver the specific gene product to any desired tissue.

Most importantly, hematopoietic tissues contain stem cells, whichpossess extensive (perhaps unlimited) capacities for self-renewal. Thisimplies that if genetic material were stably transduced into these stemcells, then upon reinfusion of the hematopoietic tissue, these alteredstem cells can expand and repopulate the marrow with cells that expressthe new gene. This leads to long-lasting, perhaps lifelong delivery ofthe desired gene product. Similarly, successful stable gene transferinto stem cells located in other tissues, or into embryonic stem cells,likewise leads to long-lasting gene product delivery.

Successful hematopoietic stem cell gene therapy has broad application,to both diseases specific to the hematopoietic system and to other organsystem diseases. Within the hematopoietic system, both inherited andacquired diseases can be treated by stem cell gene therapy. For example,hemoglobin deficiencies such as α and β Thalessemias could be treated bythe insertion of the gene coding for the globin α or β chain, togetherwith regulatory sequences that confer high level tissue specificerythrocytes (see, Grosveld et al, Cell (1987) 51:975-986). Similarly,sickle cell anemia could be corrected by the genetic insertion of thefetal globin gene into hematopoietic stem cells, as the regulatedexpression of high levels of fetal hemoglobin are sufficient to preventsickling in red cells despite the copresence of sickle hemoglobin (see,Sunshine et al, J. Molec. Biol. (1979) 133:435).

Genetic disease of neutrophils caused by functional proteindeficiencies, such as leukocyte adhesion deficiency (LAD) or chronicgranulomatous disease (CGD) could be treated by the genetic insertion ofthe gene encoding the defective or absent gene, along with regulatoryDNA sequences that confer high level, tissue specific expression intohematopoietic stem cells (see, Wilson et al, Science (1990)248:1413-1416). Genetic diseases involving platelets, such as vonWillebrands' Disease, could be corrected by the genetic insertion of thegene encoding, e.g. von Willebrands' Factor, along with sequences whichpermit its expression and secretion.

The particular suitability of hematopoietic stem cell gene therapy forthe replacement of congenitally deficient gene products is particularlyevident in the treatment of lymphocyte immunodeficiency diseases, suchas severe combined immunodeficiency due to adenosine deaminasedeficiency. Retroviral gene therapy of circulating T cells with the ADAgene has been found to be successful at reducing the clinicalimmunodeficiency experienced by these patients, but the effects are onlytemporary because the transfected T lymphocytes have a finite life spanin vivo (see, Kasid et al, Proc. Nat. Acad. Sci. (USA) (1990)87:473-477, or Culver et al Proc. Nat. Acad. Sci. (USA), (1991)88:3155-3159). If, however, the gene could be successfully transfectedinto hematopoietic stem cells, then all of the T cells which arose fromthese stem cells would contain and express the ADA gene. Therefore,since the transfected stem cells would persist and proliferate for thelife of the patient, the T cell ADA deficiency would be permanentlytreated by a single gene transfer stem cell treatment (see, Wilson etal, Proc. Natl. Acad. Sci., (U.S.A.) (1990) 87:439-443).

In addition to treating inherited enzymatic abnormalities of thehematopoietic system, stem cell gene therapy could be useful forprotecting stem cells and their progeny from toxic exogenous agents suchas viruses or chemotherapy. For example, gene transfer of DNA sequencesencoding the TAR binding site of the HIV TAT transactivating factor havebeen shown to protect T cells from spreading infection by the HIV virus(see, Sullenger et al, Cell (1990) 63:601-608). Stable transaction ofthese sequences into hematopoietic stem cells would result in a pool ofT cells, all arising from these stem cells, which were relatively orabsolutely resistant to the spread of HIV.

Similarly, successful transfection of the genes encoding the multi-drugresistance gene (MDR) or the methotrexate resistance gene into humanbone marrow stem cells would create stem cells which were relativelyresistant to the effects of cancer chemotherapy. Following autologousbone marrow transplantation with these genetically manipulated cells,patients would be able to tolerate chemotherapy with the agents to whichtheir stem cells were protected with suffering the profound bone marrowsuppression commonly caused by these anti-cancer drugs. This wouldenable patients to receive more effect doses of cancer chemotherapy withless toxicity.

One can readily envision that hematopoietic stem cell gene therapy willalso be useful for acquired hematopoietic disease such as leukemia,lymphoma and aplastic anemia. Once the genetic causes of these diseasesis discovered, insertion of a gene whose product either overcomes thatof the abnormal gene in the cell or corrects it directly (perhaps bysplicing out and replacing the gene) would correct the abnormality.

On a broader level, however, hematopoietic stem cell gene therapy can beuseful for the treatment of diseases outside the hematopoietic system aswell. Gene transfer of DNA sequences carrying therapeutic solubleproteins could give rise to mature blood cells which permanentlysecreted the desired amounts of a therapeutic molecule. By way ofexamples, this approach could be useful for the treatment of, e.g.,diabetes mellitus by the insertion of DNA sequences for insulin alongwith regulatory DNA sequences that controlled the proper expression ofthe transfected insulin gene, perhaps in response to elevated plasmaglucose levels. Systemic hypertension could be treated by geneticinsertion of stem cells with DNA sequences encoding secretory peptideswhich act as competitive inhibitors to angiotensin converting enzyme, tovascular smooth muscle calcium channels, or to adrenergic receptors.Alzheimer's disease could possibly be treated by. genetic insertion intostem cells of DNA sequences encoding enzymes which break down amyloidplaques within the central nervous system.

The many applications of gene therapy, particularly via stem cellgenetic insertion, are thus well known and have been extensivelyreviewed (see, Boggs et al, supra, Kohn et al, supra, Lehn, supra,and/or Verma et al, supra). There are indeed increasing examples of somesuccess in achieving therapeutic gene transfer into differentiated humanstem cells, as described for example in T lymphocytes (see, Kalsd et al,Proc. Nat. Acad. Sci. (U.S.A.), (1990) 87:473-477, Culver et al, Proc.Nat. Acad. Sci. (U.S.A.) (1991) 88:3155-3159).

Unfortunately, achieving (stable) gene transfer into human stem cellshas not been accomplished prior to the present invention. While severalgroups have demonstrated the feasibility of retroviral mediated genetransfer into human hematopoietic cells, human primitive hematopoieticstem cells have not been successfully transfected. This is in sharpcontrast to experiments in the mouse, in which some level ofretrovitally mediated gene transfer into hematopoietic stem cells hasbeen possible (see, Wilson et al, Pro. Nat. Acad. Sci. (USA) (1990)87:439-443).

The major impediment to achieving successful human hematopoietic stemcell gene therapy has been the inability to insert genes into humanhematopoietic cells under conditions in which the stem cells aredividing and proliferating. Successful stable gene insertion into atarget cell requires that the target cell undergo at least one round ofcell division. Thus if stem cells are not dividing in the presence ofthe desired genetic material, the material will not be stably insertedinto the stem cells. Prior to the development of the present invention,no system existed which supported the ex vivo division and proliferationof human stem and no successful genetic transformation of human stemcells has been possible.

There is therefore a considerable need for methods and compositions forthe ex vivo replication and stable genetic transformation of human Stemcells and for the optimization of human hematopoietic progenitor cellcultures, particularly in light of the great potential for stem cellexpansion, progenitor cell expansion, and gene therapy offered by thesesystems. Unfortunately, to date, attempts to achieve such results havebeen disappointing.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this Invention to provide novel methods,including culture media conditions, for the ex vivo replication andstable genetic transformation of human stem cells.

It is another object of this invention to provide novel methods,including culture media conditions, for the optimization (i) of humanhematopoietic progenitor cell cultures and (ii) of obtaining stablygenetically transformed human hematopoietic progenitor cells.

The present invention is based on the inventors' discovery of novelmethods, including culture media conditions, which provide for ex vivohuman stem cell division and stable genetic transformation and/or theoptimization of human hematopoietic progenitor cell cultures. Thesemethods rely on culturing human stem cells and/or human hematopoieticprogenitor cells in a liquid culture medium which is replaced,preferably perfused, either continuously or periodically, at a rate of 1milliliter (ml) of medium per ml of culture per about 24 to about 48hour period, and removing metabolic products and replenishing depletednutrients while maintaining the culture under physiologically acceptableconditions. In a particularly preferred embodiment of the presentinvention, the above medium replacement rate is used in conjunction withthe addition of hematopoietic growth factors to the rapidly exchangedculture medium.

The inventors have discovered that the increased medium exchange rateused in accordance with the present invention, with the optionaladdition of hematopoietic growth factors to the rapidly exchangedculture medium, surprisingly (1) supports cultures in which human stemcells proliferate over extended periods of time of at least 5 months,(2) supports cultures in which human hematopoietic progenitor cells areproduced by division and differentiation of human stem cells throughextended culture periods of at least 5 months, and (3) stimulates theincreased metabolism of and GM-CSF secretion from human stromal cells,including human bone marrow stromal cells. The present inventionprovides, for the first time, human stem cell survival and proliferationin culture.

The present invention also provides an ex vivo culture system whichsupports the continuous proliferation of human stem cells to allow thesuccessful insertion of genetic material into the human stem cells,resulting in the creation of stably genetically transformed human stemcells. This embodiment of the invention can be used for the transfer ofany genetic material that can be engineered into a recombinantretrovirus, or any other gene transfer vector that requires celldivision. Genetically modified human stem cells produced in this mannercan be applied to a wide variety of clinical diseases, as describedsupra.

The invention also provides methods for enhancing the efficiency ofgenetic transfer into human hematopoietic progenitor cells, togetherwith providing stably genetically transformed human stem cells and/orstably genetically transformed human hematopoietic progenitor cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The advantages of the present invention may be observed whenever thepresent invention is applied to any standard system for liquid humanstem and/or progenitor cell culture, such as those found inhematopoietic culture(s). By the use of the rapid medium exchange ratesused in accordance with the present invention, with the optionaladdition of supplementary hematopoietic growth factors to the culture,the inventors have surprisingly discovered that one is able to makestandard systems for liquid human hematopoietic cultures, which comprisecultures performed in the presence or absence of animal sera or plasmas,including horse, calf, fetal calf, or human serum, perform in aqualitatively superior manner.

Human liquid hematopoietic cultures which may be used in accordance withthe invention can be performed at cell densities of from 10⁴ to 10⁹cells per ml of culture, using standard known medium components such as,for example, IMDM, MEM, DMEM, RPMI 1640, Alpha Medium or McCoy's Medium,which can use combinations of serum albumin, cholesterol and/orlecithin, selenium and inorganic salts. As known, these cultures may besupplemented with corticosteroids, such as hydrocortisone at aconcentration of 10⁻⁴ to 10⁻⁷ M, or other corticosteroids at equalpotent dose, such as cortisone, dexamethasone or solumedrol. Thesecultures are typically carried out at a pH which is roughly physiologic,i.e. 6.9 to 7.4. The medium is typically exposed to an oxygen-containingatmosphere which contains from 4 to 20 vol. percent oxygen, preferably 6to 8 vol. percent oxygen.

Using these standard culture techniques, the cell mass used may beenriched, by any desired amount, such as by up to 10³ fold or more,either for stem cell content or for hematopoietic progenitor cellcontent. Different known methods may be used to achieve this enrichment,corresponding either to a negative selection method or a positiveselection method. For example, in accordance with the negative selectionmethod, mature cells are removed using immunological techniques, e.g.,labelling non-progenitor, non-stem cells with a panel of mouseanti-human monoclonal antibodies, then removing the mouseantibody-coated cells by adherence to rabbit-anti-mouse Ig-coatedplastic dishes. See e.g., Emerson et al, J. Clin. Invest. (1985)76:1286-1290.

The present invention relies on a fundamental alteration of theconditions of liquid human bone marrow cultures under any of the aboveconditions; rapid replacement of the nutrient medium. Standard cultureschedules call for medium and serum to be exchanged weekly, either as asingle exchange performed weekly or a one-half medium and serum exchangeperformed twice weekly. In accordance with the present invention, thenutrient medium of the culture is replaced, preferably perfused, eithercontinuously or periodically, at a rate of about 1 ml per ml of cultureper about 24 to about 48 hour period, for cells cultured at a density offrom 2×10⁶ to 1×10⁷ cells per ml. For cell densities of from 1×10⁴ to2×10⁶ cells per ml the same medium exchange rate may be used. For celldensities higher than 10⁷ cells per ml, the medium exchange rate may beincreased proportionally to achieve a constant medium and serum flux percell per unit time.

Replacement of the nutrient medium in accordance with the invention maybe carried out in any manner which will achieve the result of replacingthe medium, e.g., by removing an aliquot of spent culture medium andreplacing it with a fresh aliquot. The flow of the aliquot being addedmay be by gravity, by pump, or by any other suitable means. The flow maybe in any direction or multiplicity of directions, depending upon theconfiguration and packing of the culture. Preferably, the new medium isadded to the culture in a manner such that it contacts the cell mass.Most preferably, it is added the culture in a manner mimicking in vivoperfusion, i.e., it is perfused through at least part of the cell massand up to the whole cell mass.

Another, optional but important, embodiment of the present invention,resides in the addition of hematopoietic growth factors, includingsynthetic hematopoietic growth factors, to the rapidly exchangedcultures. In a particularly preferred aspect of this embodiment, thecytokines IL-3 and GM-CSF are both added, together, to the medium at arate of from 0.1 to 100 ng/ml/day, preferably about 0.5 to 10 ng/ml/day,most preferably 1 to 2 ng/ml/day. Epo may be added to the nutrientmedium in an amount of from 0.001 to 10 U/ml/day, preferably 0.05 to0.15 U/ml/day. Mast cell growth factor (MCGF, c-kit ligand, Steelfactor), may be added to the medium in an amount of from 1 to 100ng/ml/day, preferably 10 to 150 ng/ml/day. IL-1 (α or β) may also beadded in an amount of from 10 to 100 units/ml per 3 to 5 day period.Additionally, IL-6, G-CSF, basic fibroblast growth factor, IL-7, IL-8,IL-9, IL-10, IL-11, PDGF, or EGF to be added, at a rate of from 1 to 100ng/ml/day.

The inventors have discovered that when IL-3, GM-CSF and Epo are used asdescribed above one obtains lineage specific development of red bloodcells. Alternatively, when IL-3 and GM-CSF, with or without IL-6 orG-CSF, are used, the culture preferentially produce granulocytes. Theinventors also observed that with the cultures of the invention T and Blymphocytes are lost over time.

The metabolic product level in the medium is normally maintained withina particular range. Glucose concentration is usually maintained in therange of about 5 to 20 mM. Lactate concentration is usually maintainedbelow 35 mM. Glutamine concentration is generally maintained in therange of from about 1 to 3 mM. Ammonium concentration is usuallymaintained below about 2.4 mM. These concentrations can be monitored byeither periodic or on-line continuous measurements using known methods.See, e.g., Caldwell et al, J. Cell. Physiol. (1991) 147:344-353.

The cells which may be cultured in accordance with the present inventionmay be any human stem cells or human stem cell-containing cellular mass,including human peripheral blood mononuclear cells, human bone marrowcells, human fetal liver cells, embryonic stem cells and/or human cordblood cells. Each of these cell masses contains human stem cells and/orhuman hematopoietic progenitor cells. Other cellular masses containinghuman stem cells may also be used in accordance with the invention,including any human stem cell found in human bone marrow.

In a preferred embodiment of the invention, the cell culture may beenriched to augment the human stem cell content of the cell mass. Suchenrichment may be achieved as described above, and, when used inaccordance with the invention, provides the first useful means forgenetic therapy via gene transfer into human stem cells, including humanstem cells present in human bone marrow and human bone marrow stemcells. Stem cells present in human bone marrow are cells obtainable fromhuman bone marrow, peripheral blood, fetal liver, or human cord blood.

Generally, in this embodiment, a packaging cell line infected with aretrovirus, or a supernatant obtained from such a packaging cell lineculture, or another gene transfer vector, is added to human stem cellscultured in accordance with the invention to obtain stably geneticallytransformed human stem cells. The present invention provides increasedlevels of stem cell and human hematopoietic progenitor cell replication,whereas, by contrast, prior cultures provided only for humanhematopoietic progenitor cell replication at a decreasing rate (i.e.,decaying cultures). The present culture system provides, for the firsttime, expansion of cells in culture, which is required for retroviralinfection of cells. Earlier systems in which retroviral infection wascarried out on decaying cultures provided no infection of earlier cells.The present invention, particularly when it is practiced together withan enriched stem cell pool, and even more particularly when it ispracticed still further with the use of hematopoietic growth factors,including synthetic growth factors, provides a very effective means forobtaining stem cell infection in vitro.

The inventors have discovered that addition of supernatants containingrecombinant retroviruses to the cultures results in the introduction ofthe viruses and the genes they carry into human (hematopoietic) stemcells. The progenitor cells which arise by division and differentiationfrom these stem cells, and the mature blood cells which arise fromfurther division and differentiation of these progenitor cells, containthe transfected DNA throughout the period of the hematopoietic culturein vitro. The inventors have observed that when the retrovirus is onlyadded at the beginning period of the culture, they obtain transfectedprogenitors and mature blood cells which can arise only from stem cellspresent, proliferating and stably, genetically transformed in thebeginning of the culture, because no retrovirally infected cell caninfect an adjacent cell. Progenitor cells and more mature cellscontaining the desired genetic material have accordingly received thegene from the more primitive stem cells genetically transformed duringthe initial retroviral infection period.

In a preferred embodiment of this aspect of the invention, humanhematopoietic cells, either isolated from bone marrow, peripheral blood,fetal liver, or umbilical cord blood, are first enriched for thepresence of stem cells by removing more mature blood cells. This isaccomplished by incubating the hematopoietic cells with murinemonoclonal antibodies recognizing epitopes on mature blood cells andbone marrow precursor cells but not stem cells, and then removing thelabelled cells by immunoadherence to a rabbit-anti-mouse-Igimmunoadsorbent surface. The resultant lineage negative (Lin⁻) cells arethen cultured in the presence of a retrovirus or other gene transfervector in accordance with the invention. Preferably the culture iscarried out in the presence of GM-CSF (preferably 1 mg/ml/day) and IL-3(preferably 1 mg/ml/day) with or without IL-1 (preferably 50 U/ml/4 dayperiod), with or without c-kit ligand (Mast cell growth factor)(preferably 10 ug/ml/day).

The retroviral infection may be performed by either including into theculture medium, supernatants (e.g., 5 to 20% vol/vol) produced byretroviral packaging cell lines infected with recombinant retrovirus,during the first 2 to 21, preferably 10 to 14 days of the culture, or byculturing the Lin⁻ cells directly over the infected retroviral packaginglines themselves, or by both.

Preferably, retroviral supernatants are used, and the period ofincubation in the presence of virus is 12 to 16 days. Also preferably,the packaging cell lines are grown to near confluency, the mediumexchanged, and the cell lines further incubated for 12 to 15 hours. Themedium is then collected and used in the transfection of the human stemcells. However, this protocol is not strictly required and anysupernatant produced by a retroviral packaging cell line may be used.Any (known) retroviral packaging cell lines may be used in accordancewith the invention and cultured in accordance with any known protocol(see, e.g., Wilson et al, Science (1990) 248:1413-1416 and/or Sullengeret al, Cell (1990) 63:601-608). Illustrative packaging cell linesinclude NIH 3T3 cells and renal carcinoma cell line 5637.

Any gene which is inserted into a recombinant retrovirus together withsuitable promoter and enhancer elements that permit its expression canbe incorporated into human stem and hematopoietic progenitor cells. Theinvention provides for the first time conditions that permit stem cellsurvival and proliferation in these cultures, permitting the creation ofstably transfected, genetically modified human hematopoietic stem cellsin these cultures. The terms "stably transformed" and "stablytransfected" are used in this text to designate incorporation ofexogenous DNA into the human stem cell chromosome(s), made possible bythe present invention because it permits exposing dividing human stemcells ex vivo to such exogenous DNA.

In accordance with the present invention one obtains cultures in whichhuman hematopoietic progenitor cells are produced by division anddifferentiation from human stem cells throughout a culture period of atleast five months. That is, one obtains a culture which supports stemcell survival and proliferation in culture.

Data obtained by the inventors indicates that medium perfusion rate is avery significant variable in determining the behavior of ex vivo humanbone marrow cultures. This data showed that when the medium exchangerate was increased from the traditional once per week Dexter rate to adaily medium exchange rate of 7 volumes per week, a significant effecton ex vivo hematopoiesis is obtained. In experiments carried out by theinventors, all cultures displayed a significant loss of cells during thefirst 3 to 4 weeks. Following this decay, the cultures stabilized andthe effect of a medium perfusion rate became more pronounced.

A 3.5 per week medium exchange rate led to the most prolific culturesand also to cultures of greatest longevity in terms of progenitor cellproduction. Of particular note, during weeks 4 to 10, the biweeklynumber of nonadherent cells produced was actually stable or increasing.

Over the entire course of the cultures, the cumulative number of cellsproduced after week 3.5 was almost three-fold greater than that which isproduced under the traditional Dexter culture protocol. Further, stableproduction of progenitor Cells is maintained until week 18.

Human stromal cells, such as stromal cells found in human bone marrow,may or may not be present in the cultures of the invention. In typicalcultures, stromal cells are present in the cell culture in an amount ofapproximately 10⁻³ to 10⁻¹ (stromal cells/total cells).

In another aspect of the invention, the inventors discovered that thecultures of the invention surprisingly provide increased metabolism andGM-CSF and IL-6 secretion from human bone marrow stromal cells. Whereasno GM-CSF is detected in human bone marrow stromal cells supernatant,rapid medium exchange in accordance with the invention stimulates humanbone marrow stromal cells to secrete 300 centigrams/ml/day to 200picograms/ml/day of GM-CSF. Secretion of IL-6 by human bone marrowstromal cells is also increased by rapid medium exchange in accordancewith the invention from 1 to 2 ng/ml/day to 2 to 4 ng/ml/day. Thisincrease is observed both when only the rapid medium exchange rate ofthe invention is used, and when the rapid exchange rate together withthe addition of hematopoietic growth factors is used. On the basis ofdata obtained by the inventors, the effect of the rapid medium exchangerates of the invention on human stromal cell production of cytokinesshould be observed with human stromal cells in any complex tissueculture system.

Illustratively, the medium used in accordance with the invention maycomprise three basic components. The first component is a mediacomponent comprised of IMDM, MEM, DMEM, RPMI 1640, Alpha Medium orMcCoy's Medium, or an equivalent known culture medium component. Thesecond is a serum component which comprises at least horse serum orhuman serum and may optionally further comprise fetal calf serum,newborn calf serum, and/or calf serum. The third component is acorticosteroid, such as hydrocortisone, cortisone, dexamethasome,solumedrol, or a combination of these, preferably hydrocortisone.

The compositional make up of various media which can be used are setforth below.

    ______________________________________                                        Iscove's Modified Dulbecco's Media (IMDM).sup.1,2,3                                             380-2440 430-2200                                                             1X Liquid                                                                              Powder                                             COMPONENT         mg/L     mg/L                                               ______________________________________                                        INORGANIC SALTS:                                                              CaCl.sub.2 (anhyd.)                                                                             165.00   165.00                                             KCl               330.00   330.00                                             KNO.sub.3         0.076    0.076                                              MgSO.sub.4 (anhyd.)                                                                             97.67    97.67                                              NaCl              4505.00  4505.00                                            NaHCO.sub.3       3024.00  --                                                 NaH.sub.2 PO.sub.4.H.sub.2 O.sup.a                                                              125.00   125.00                                             Na.sub.2 SeO.sub.3 5H.sub.2 O                                                                   0.0173   0.0173                                             OTHER COMPONENTS:                                                             D-Glucose         4500.00  4500.00                                            Phenol red        15.00    15.00                                              HEPES             5958.00  5958.00                                            Sodium pyruvate   110.00   110.00                                             AMINO ACIDS:                                                                  L-Alanine         25.00    25.00                                              L-Asparagine.H.sub.2 O                                                                          28.40    28.40                                              L-Arginine.HCl    84.00    84.00                                              L-Aspartic acid   30.00    30.00                                              L-Cystine.2HCl    91.24    91.24                                              L-Glutamic acid   75.00    75.00                                              L-Glutamine       584.00   584.00                                             Glycine           30.00    30.00                                              L-Histidine.HCl.H.sub.2 O                                                                       42.00    42.00                                              L-Isoleucine      105.00   105.00                                             L-Leucine         105.00   105.00                                             L-Lysine.HCl      146.00   146.00                                             L-Methionine      30.00    30.00                                              L-Phenylalanine   66.00    66.00                                              L-Proline         40.00    40.00                                              L-Serine          42.00    42.00                                              L-Threonine       95.00    95.00                                              L-Tryptophan      16.00    16.00                                              L-Tyrosine.2Na.2H.sub.2 O                                                                       103.79   103.79                                             L-Valine          94.00    94.00                                              VITAMINS:                                                                     Biotin            0.013    0.013                                              D-Ca pantothenate 4.00     4.00                                               Choline chloride  4.00     4.00                                               Folic acid        4.00     4.00                                               i-Inositol        7.20     7.20                                               Niacinamide       4.00     4.00                                               Pyridoxal.HCl     4.00     4.00                                               Riboflavin        0.40     0.40                                               Thiamine.HCl      4.00     4.00                                               Vitamin B.sub.12  0.013    0.013                                              ______________________________________                                         1. Dulbecco, R. and Freeman, G. (1959) Virology 8, 396. Smith, J. D.,         Freeman, G., Vogt, M., and Dulbecco, R. (1960) Virology 12, 185, Tissue       Culture Standards Committee, In Vitro 6:2, 93.                                2. Iscove, N. N. and Melchers, F., J. Experimental Medicine 147,923.          a. Values shown are in conformance with Tissue Culture Standards              Committee, In Vitro (1970) 9:6.                                               3. Iscove, N. N., personal communication.                                

                                                                                     - Dulbecco's.sup.1 Modified Eagle Media (D-MEM)                                 320-1885 380-2320 430-1600 320-1965 380-2430 430-2100 430-2800               430-3000 320-1960 320-1970 320-1995 430-3700 320-1968 430-3000                   1X Liquid 1X Liquid Powder 1X Liquid 1X Liquid Powder Powder Powder 1X       Liquid 1X Liquid 1X Liquid Powder 1X Liquid Powder                              COMPONENT mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L         mg/L mg/L                                                                       INORGANIC SALTS:                                                               CaCl.sub.2 (anhyd.) 200.00 200.00 200.00 200.00 200.00 200.00 200.00          200.00 200.00 200.00 200.00 200.00 200.00 200.00                                Fe(NO.sub.3).sub.3.9H.sub.2 O 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10         0.10 0.10 0.10 0.10 0.10                                                        MgSO.sub.4 (anhyd.) 400.00 400.00 400.00 400.00 400.00 400.00 400.00          400.00 400.00 400.00 400.00 400.00 400.00 400.00                                KCl -- -- 97.67 -- -- 97.67 97.67 97.67 -- -- -- 97.67 -- 97.67                        MgSO.sub.4.7H.sub.2 O 200.00 200.00 -- 200.00 200.00 -- -- --         200.00 200.00 200.00 -- 200.00 --                                               NaCl 6400.00 4750.00 6400.00 6400.00 4750.00 6400.00 6400.00 6400.00          6400.00 6400.00 6400.00 4750.00 6400.00 6400.00                                 NaCO.sub.3 3700.00 3700.00 -- 3700.00 3700.00 -- -- -- 3700.00 3700.00        3700.00 -- 3700.00 --                                                           Na.sub.2 PO.sub.4.H.sub.2 O.sup.a 125.00 125.00 125.00 125.00 125.00          125.00 125.00 125.00 125.00 125.00 125.00 125.00 125.00 125.00                  OTHER COMPONENTS:                                                              D-Glucose 1000.00 1000.00 1000.00 4500.00 4500.00 4500.00 4500.00             4500.00 4500.00 4500.00 4500.00 4500.00 4500.00 --                              Phenol red 15.00 15.00 15.00 15.00 15.00 15.00 15.00 -- 15.00  15.00          15.00 15.00 --                                                                  HEPES -- 5958.00 -- -- 5958.00 -- -- -- -- -- -- 5958.00 -- --                 Sodium pyruvate 110.00 110.00 110.00 -- -- -- 110.00 -- -- -- 110.00 --       -- --                                                                           AMINO ACIDS:                                                                   L-Arginine-HCl 84.00 84.00 84.00 84.00 84.00 84.00 84.00 84.00 84.00          84.00 84.00 84.00 84.00 84.00                                                   L-Cystine 48.00 48.00 -- 48.00 48.00 -- -- -- 48.00 48.00 48.00 --            48.00 --                                                                        L-Cystine.2HCl -- -- 62.57 -- -- 62.57 62.57 62.57 -- -- -- 62.57 --          62.57                                                                           L-Glutamine 584.00 584.00 584.00 584.00 584.00 584.00 584.00 584.00 --        -- 584.00 584.00 584.00 --                                                      Glycine 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00           30.00 30.00 30.00 30.00                                                         L-Histidine.HCl.H.sub.2 O 42.00 42.00 42.00 42.00 42.00 42.00 42.00           42.00 42.00 42.00 42.00 42.00 42.00 42.00                                       L-Isoleucine 105.00 105.00 105.00 105.00 105.00 105.00 105.00 105.00          105.00 105.00 105.00 105.00 105.00 105.00                                       L-Leucine 105.00 105.00 105.00 105.00 105.00 105.00 105.00 105.00             105.00 105.00 105.00 105.00 105.00 105.00                                       L-Lysine-HCl 146.00 146.00 146.00 146.00 146.00 146.00 146.00 146.00          146.00 146.00 146.00 146.00 146.00 146.00                                       L-Methionine 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 --         30.00 30.00 30.00 30.00                                                         L-Phenylalanine 66.00 66.00 66.00 66.00 66.00 66.00 66.00 66.00 66.00         66.00 66.00 66.00 66.00 66.00                                                   L-Serine 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00          42.00 42.00 42.00 42.00                                                         L-Threonine 95.00 95.00 95.00 95.00 95.00 95.00 95.00 95.00 95.00 95.00       95.00 95.00 95.00 95.00                                                         L-Tryptophan 16.00 16.00 16.00 16.00 16.00 16.00 16.00 16.00 16.00            16.00 16.00 16.00 16.00 16.00                                                   L-Tyrosine 72.00 72.00 -- 72.00 72.00 -- -- -- 72.00 72.00 72.00 --           72.00 --                                                                        L-Tyrosine.2Na.2H.sub.2 O -- -- 103.79 -- -- 103.79 103.79 103.79 -- --       -- 103.79 -- 103.79                                                             L-Valine 94.00 94.00 94.00 94.00 94.00 94.00 94.00 94.00 94.00 94.00          94.00 94.00 94.00 94.00                                                         VITAMINS:                                                                      D-Ca pantothenate 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00           4.00 4.00 4.00 4.00                                                             Choline chloride 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00       4.00 4.00 4.00                                                                  Folic acid 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00        4.00 4.00                                                                       i-Inositol 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20        -- 7.20                                                                         Niacinamide 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00       4.00 4.00                                                                       Pyridoxal.HCl 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00          4.00 4.00 4.00                                                                  Riboflavin 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40        0.40 0.40                                                                       Thiamine.HCl 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00           4.00 4.00 4.00                                                                 .sup.1 Dulbecco, R. and Freeman, G. (1959) Virology 8, 396. Smith, J. D.,      Freeman, G., Vogt, M., and Dulbecco, R. (1960) Virology 12, 185. Tissue        Culture Standards Committee, In Vitro 6:2, 93.                                 .sup.a Values shown are in conformance with the Tissue Culture Standards       Committee, In Vitro (1970) 9:6.                                           

                                                                                     - Minimum Essential Media (MEM).sup.1                                           20-2561.sup.2 410-2000.sup.2 320-2571.sup.2 410-1900.sup.1 320-2570          320-1090 380-2360 330-1430 410-1700 320-1890 320-1096 410-2400 320-1097        410-2500                                                                         1X Liquid Powder 1X Liquid Powder 1X Liquid 1X Liquid 1X Liquid 10X          Liquid Powder 1X Liquid 1X Liquid Powder 1X Liquid Powder                       COMPONENT mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L         mg/L mg/L                                                                       INORGANIC SALTS:                                                               CaCl.sub.2 (anhyd.) 200.00 200.00 200.00 200.00 200.00 200.00 200.00          2000.00 200.00 200.00 200.00 200.00 200.00 200.00                               KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00 4000.00 400.00           400.00 400.00 400.00 400.00 400.00                                              MgSO.sub.4 (anhyd.) -- 97.67 -- 97.57 -- -- -- -- 97.67 -- -- 97.67 --        97.67                                                                           MgSO.sub.4.7H.sub.2 O 200.00 -- 200.00 -- 200.00 200.00 200.00 2000.00        -- 200.00 200.00 -- 200.00 --                                                   NaCl 6800.00 6800.00 6800.00 6800.00 6800.00 6800.00 6350.00 68000.00         6800.00 6800.00 6800.00 6800.00 6800.00 6800.00                                 NaHCO.sub.3 2200.00 -- 2200.00 -- 2200.00 2200.00 2200.00 -- -- 2200.00       1500.00 -- 2200.00 --                                                           NaH.sub.2 PO.sub.4.H.sub.2 O.sup.a 140.00 140.00 140.00 140.00 140.00         140.00 140.00 1400.00 140.00 140.00 140.00 140.00 -- --                         OTHER COMPONENTS:                                                              D-Glucose 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00             10000.00 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00                        HEPES -- -- -- -- -- -- 5958.00 -- -- -- -- -- -- --                           Lipic acid 0.20 0.20 0.20 0.20 -- -- -- -- -- -- -- -- -- --                   Phenol red 10.00 10.00 10.00 10.00 10.00 10.00 10.00 100.00 6.00 10.00        10.00 10.00 10.00 10.00                                                         Sodium pyruvate 110.00 110.00 110.00 110.00 -- -- -- -- -- -- -- -- --        --                                                                              Sodium succinate -- -- -- -- -- -- -- -- 100.00 -- -- -- -- --                 Succinic acid -- -- -- -- -- -- -- -- 75.00 -- -- -- -- --                     AMINO ACIDS:                                                                   L-Alanine 25.00 25.00 25.00 25.00 -- -- -- -- -- -- -- -- -- --                        L-Arginine 105.00 -- 105.00 -- -- -- -- -- -- -- -- -- -- --              L-Arginine.HCl -- 126.64 -- 126.64 126.00 126.00 126.00 1260.00            126.00 126.00 126.00 126.00 126.00 126.00                                       L-Asparagine.H.sub.2 O 50.00 50.00 50.00 50.00 -- -- -- -- -- -- -- --        -- --                                                                           L-Aspartic acid 30.00 30.00 30.00 30.00 -- -- -- -- -- -- -- -- -- --            L-Cystine 24.00 -- 24.00 -- 24.00 24.00 24.00 240.00 -- 24.00 -- --         24.00 --                                                                        L-Cystine.2HCl -- 31.28 -- 31.28 -- -- -- -- 31.00 -- 31.00 31.29 --          31.29                                                                           L-Cysteine.HCl.H.sub.2 O 100.00 100.00 100.00 100.00 -- -- -- -- -- --        -- -- -- --                                                                     L-Glutamic acid 75.00 75.00 75.00 75.00 -- -- -- -- -- -- -- -- -- --            L-Glutamine 292.00 292.00 292.00 292.00 292.00 -- -- -- -- -- --            292.00 292.00 292.00                                                            Glycine 50.00 50.00 50.00 50.00 -- -- -- -- -- -- -- -- -- --                  L-Histidine 31.00 -- 31.00 -- -- -- -- -- -- -- -- -- -- --                    L-Histidine.HCl.H.sub.2 O -- 42.00 -- 42.00 42.00 42.00 42.00 420.00          42.00 42.00 42.00 42.00 42.00 42.00                                             L-Isoleucine 52.40 52.40 52.40 52.40 52.00 52.00 52.00 520.00 52.00           52.00 52.00 52.00 52.00 52.00                                                   L-Leucine 52.40 52.40 52.40 52.40 52.00 52.00 52.00 520.00 52.00 --           52.00 -- 52.00 52.00                                                            L-Lysine 58.00 -- 58.00 -- -- -- -- -- -- -- -- -- -- --                       L-Lysine.HCl -- 72.50 -- 72.50 72.50 72.50 72.50 725.00 72.50 72.50           72.50 -- 72.50 72.50                                                            L-Methionine 15.00 15.00 15.00 15.00 15.00 15.00 15.00 150.00 15.00           15.00 15.00 -- 15.00 15.00                                                      L-Phenylalanine 32.00 32.00 32.00 32.00 32.00 32.00 32.00 320.00 32.00        32.00 32.00 32.00 32.00 32.00                                                   L-Proline 40.00 40.00 40.00 40.00 -- -- -- -- -- -- -- -- -- --                        L-Serine 25.00 25.00 25.00 25.00 -- -- -- -- -- -- -- -- -- --          L-Threonine 48.00 48.00 48.00 48.00 48.00 48.00 48.00 480.00 48.00           48.00 48.00 48.00 48.00 48.00                                                   L-Tryptophan 10.00 10.00 10.00 10.00 10.00 10.00 10.00 100.00 10.00           10.00 10.00 10.00 10.00 10.00                                                   L-Tyrosine 36.00 -- 36.00 -- 36.00 36.00 36.00 360.00 36.00 36.00 -- --       36.00 --                                                                        L-Tyrosine.2Na.2H.sub.2 O -- 51.90 -- 51.90 -- -- -- -- -- -- 51.90           51.90 -- 51.90                                                                  D-Valine -- -- -- -- 92.00 -- -- -- -- -- -- -- -- --                          L-Valine 46.00 46.00 46.00 46.00 -- 46.00 46.00 460.00 46.00 46.00            46.00 46.00 46.00 46.00                                                         VITAMINS:                                                                      L-Ascorbic acid 50.00 50.00 50.00 50.00 -- -- -- -- -- -- -- -- -- --            Biotin 0.10 0.10 0.10 0.10 -- -- -- -- -- -- -- -- -- --                     D-Ca pantothenate 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00           1.00 1.00 1.00 1.00                                                             Choline bitartrate -- -- -- -- -- -- -- -- 1.80 -- -- -- -- --                 Choline cloride 1.00 1.00 1.00 1.00 1.00 1.00 1.00 10.00 1.00 1.00 1.00       1.00 1.00 1.00                                                                  Folic acid 1.00 1.00 1.00 1.00 1.00 1.00 1.00 10.00 1.00 1.00 1.00 1.00       1.00 1.00                                                                       i-Inositol 2.00 2.00 2.00 2.00 2.00 2.00 2.00 20.00 2.00 2.00 2.00 2.00       2.00 2.00                                                                       Niacinamide 1.00 1.00 1.00 1.00 1.00 1.00 1.00 10.00 1.00 1.00 1.00           1.00 1.00 1.00                                                                  Pyridoxal.HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00 10.00 1.00 1.00 1.00         1.00 1.00 1.00                                                                  Riboflavin 0.10 0.10 0.10 0.10 0.10 0.10 0.10 1.00 0.10 0.10 0.10 0.10        0.10 0.10                                                                       Thiamine.HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00 10.00 1.00 1.00 1.00          1.00 1.00 1.00                                                                  Vitamin B.sub.12 1.36 1.36 1.36 1.36 -- -- -- -- -- -- -- -- -- --                  RIBONUCLEOSIDES:                                                          Adenosine -- -- 10.00 10.00 -- -- -- -- -- -- -- -- -- --                      Cytidine -- -- 10.00 10.00 -- -- -- -- -- -- -- -- -- --                       Guanosine -- -- 10.00 10.00 -- -- -- -- -- -- -- -- -- --                      Uridine -- -- 10.00 10.00 -- -- -- -- -- -- -- -- -- --                        DEOXYTRIBONUCLEOSIDES:                                                         2' Deoxyadenosine -- -- 10.00 10.00 -- -- -- -- -- -- -- -- -- --                    2'Deoxycytidine.HCl -- -- 11.00 11.00 -- -- -- -- -- -- -- -- --        --                                                                              2' Deoxyguanosine -- -- 10.00 10.00 -- -- -- -- -- -- -- -- -- --                    Thymidine -- -- 10.00 10.00 -- -- -- -- -- -- -- -- -- --               .sup.1 Eagle, H. (1959) Science, 130, 432.                                     .sup.2 Nature, New Biology (1971) 230, 310.                                    .sup.a Original formula lists this component as NaH.sub.2                      PO.sub.4.2H.sub.2 O.                                                      

    __________________________________________________________________________    RPMI Media 1640.sup.1                                                                     320-1870                                                                           320-1875                                                                            330-2511                                                                           380-2400                                                                           430-1800                                                                           430-3200                                                                           430-3400                                                                           320-1835                                                                           320-1877                             1X Liquid                                                                          1X Liquid                                                                           10X Liquid                                                                         1X Liquid                                                                          Powder                                                                             Powder                                                                             Powder                                                                             1X Liquid                                                                          1X Liquid                COMPONENT   mg/L mg/L  mg/L mg/L mg/L mg/L mg/L mg/L mg/L                     __________________________________________________________________________    INORGANIC SALTS:                                                              Ca(NO.sub.3).sub.2.4H.sub.2 O                                                             100.00                                                                             100.00                                                                              1000.00                                                                            100.00                                                                             100.00                                                                             100.00                                                                             100.00                                                                             100.00                                                                             100.00                   KCl         400.00                                                                             400.00                                                                              4000.00                                                                            400.00                                                                             400.00                                                                             400.00                                                                             400.00                                                                             400.00                                                                             400.00                   MgSO.sub.4 (anhyd.)                                                                       --   --    --   --   48.84                                                                              48.84                                                                              48.84                                                                              --   --                       MgSO.sub.4.7H.sub.2 O                                                                     100.00                                                                             100.00                                                                              1000.00                                                                            100.00                                                                             --   --   --   100.00                                                                             100.00                   NaCl        6000.00                                                                            6000.00                                                                             60000.00                                                                           5300.00                                                                            6000.00                                                                            6000.00                                                                            5850.00                                                                            6000.00                                                                            6000.00                  NaHCO.sub.3 2000.00                                                                            2000.00                                                                             --   2000.00                                                                            --   --   --   2000.00                                                                            2000.00                  Na.sub.2 HPO.sub.4 (anhyd.)                                                               --   --    --   --   800.00                                                                             800.00                                                                             800.00                                                                             --   --                       Na.sub.2 HPO.sub.4.7H.sub.2 O                                                             1512.00                                                                            1512.00                                                                             15120.00                                                                           1512.00                                                                            --   --   --   1512.00                                                                            --                       OTHER COMPONENTS:                                                             D-Glucose   2000.00                                                                            2000.00                                                                             20000.00                                                                           2000.00                                                                            2000.00                                                                            2000.00                                                                            2000.00                                                                            2000.00                                                                            2000.00                  Glutathione (reduced)                                                                     1.00 1.00  10.00                                                                              1.00 1.00 1.00 1.00 1.00 1.00                     HEPES       --   --    --   5958.00                                                                            --   --   5957.50                                                                            --   --                       Phenol red  5.00 5.00  50.00                                                                              5.00 5.00 --   5.00 --   5.00                     AMINO ACIDS:                                                                  L-Arginine  200.00                                                                             200.00                                                                              2000.00                                                                            200.00                                                                             200.00                                                                             200.00                                                                             200.00                                                                             200.00                                                                             200.00                   L-Asparagine                                                                              50.00                                                                              50.00 500.00                                                                             50.00                                                                              50.00                                                                              50.00                                                                              50.00                                                                              50.00                                                                              50.00                    L-Aspartic acid                                                                           20.00                                                                              20.00 200.00                                                                             20.00                                                                              20,.00                                                                             20.00                                                                              20.00                                                                              20.00                                                                              20.00                    L-Cystine   50.00                                                                              50.00 500.00                                                                             50.00                                                                              --   --   --   50.00                                                                              50.00                    L-Cystine.2HCl                                                                            --   --    --   --   65.15                                                                              65.15                                                                              65.15                                                                              --   --                       L-Glutamic acid                                                                           20.00                                                                              20.00 200.00                                                                             20.00                                                                              20.00                                                                              20.00                                                                              20.00                                                                              20.00                                                                              20.00                    L-Glutamine --   300.00                                                                              3000.00                                                                            300.00                                                                             300.00                                                                             300.00                                                                             300.00                                                                             300.00                                                                             300.00                   Glycine     10.00                                                                              10.00 100.00                                                                             10.00                                                                              10.00                                                                              10.00                                                                              10.00                                                                              10.00                                                                              10.00                    L-Histidine 15.00                                                                              15.00 150.00                                                                             15.00                                                                              15.00                                                                              15.00                                                                              15.00                                                                              15.00                                                                              15.00                    L-Hydroxyproline                                                                          20.00                                                                              20.00 200.00                                                                             20.00                                                                              20.00                                                                              20.00                                                                              20.00                                                                              20.00                                                                              20.00                    L-Isoleucine                                                                              50.00                                                                              50.00 500.00                                                                             50.00                                                                              50.00                                                                              50.00                                                                              50.00                                                                              50.00                                                                              50.00                    L-Leucine   50.00                                                                              50.00 500.00                                                                             50.00                                                                              50.00                                                                              50.00                                                                              50.00                                                                              50.00                                                                              50.00                    L-Lysine-HCl                                                                              40.00                                                                              40.00 400.00                                                                             40.00                                                                              40.00                                                                              40.00                                                                              40.00                                                                              40.00                                                                              40.00                    L-Methionine                                                                              15.00                                                                              15.00 150.00                                                                             15.00                                                                              15.00                                                                              15.00                                                                              15.00                                                                              15.00                                                                              15.00                    L-Phenylalanine                                                                           15.00                                                                              15.00 150.00                                                                             15.00                                                                              15.00                                                                              15.00                                                                              15.00                                                                              15.00                                                                              15.00                    L-Proline   20.00                                                                              20.00 200.00                                                                             20.00                                                                              20.00                                                                              20.00                                                                              20.00                                                                              20.00                                                                              20.00                    L-Serine    30.00                                                                              30.00 300.00                                                                             30.00                                                                              30.00                                                                              30.00                                                                              30.00                                                                              30.00                                                                              30.00                    L-Threonine 20.00                                                                              20.00 200.00                                                                             20.00                                                                              20.00                                                                              20.00                                                                              20.00                                                                              20.00                                                                              20.00                    L-Tryptophan                                                                              5.00 5.00  50.00                                                                              5.00 5.00 5.00 5.00 5.00 5.00                     L-Tyrosine  20.00                                                                              20.00 200.00                                                                             20.00                                                                              --   --   --   20.00                                                                              20.00                    L-Tyrosine.2Na.2H.sub.2 O                                                                 --   --    --   --   28.83                                                                              28.83                                                                              28.83                                                                              --   --                       L-Valine    20.00                                                                              20.00 200.00                                                                             20.00                                                                              20.00                                                                              20.00                                                                              20.00                                                                              20.00                                                                              20.00                    VITAMINS:                                                                     Biotin      0.20 0.20  2.00 0.20 0.20 0.20 0.20 0.20 0.20                     D-Ca pantothenate                                                                         0.25 0.25  2.50 0.25 0.25 0.25 0.25 0.25 0.25                     Choline chloride                                                                          3.00 3.00  30.00                                                                              3.00 3.00 3.00 3.00 3.00 3.00                     Folic acid  1.00 1.00  10.00                                                                              1.00 1.00 1.00 1.00 1.00 1.00                     i-Inositol  35.00                                                                              35.00 350.00                                                                             35.00                                                                              35.00                                                                              35.00                                                                              35.00                                                                              35.00                                                                              35.00                    Niacinamide 1.00 1.00  10.00                                                                              1.00 1.00 1.00 1.00 1.00 1.00                     Para-aminobenzoic acid                                                                    1.00 1.00  10.00                                                                              1.00 1.00 1.00 1.00 1.00 1.00                     Pyridoxine.HCl                                                                            1.00 1.00  10.00                                                                              1.00 1.00 1.00 1.00 1.00 1.00                     Riboflavin  0.20 0.20  2.00 0.20 0.20 0.20 0.20 0.20 0.20                     Thiamine.HCl                                                                              1.00 1.00  10.00                                                                              1.00 1.00 1.00 1.00 1.00 1.00                     Vitamin B.sub.12                                                                          0.005                                                                              0.005 0.05 0.005                                                                              0.005                                                                              0.005                                                                              0.005                                                                              0.005                                                                              0.005                    __________________________________________________________________________     .sup.1 Moore, G. E., Gerner, R. E., and Franklin, H. A. (1967) J.A.M.A.       199, 519.                                                                

    __________________________________________________________________________    McCoy's 5A Media (modified).sup.1,2,3                                                     320-6600                                                                           380-2330                                                                           430-1500                                                                           320-6608                                                                           320-6601.sup.4                                                                     320-6610                                                                           320-6620                                                                           320-6630                                   1X Liquid                                                                          1X Liquid                                                                          Powder                                                                             1X Liquid                                                                          1X Liquid                                                                          1X Liquid                                                                          1X Liquid                                                                          1X Liquid                      COMPONENT   mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L                           __________________________________________________________________________    INORGANIC SALTS:                                                              CaCl.sub.2 (anhyd.)                                                                       100.00                                                                             100.00                                                                             100.00                                                                             --   140.00                                                                             100.00                                                                             100.00                                                                             100.00                         KCl         400.00                                                                             400.00                                                                             400.00                                                                             400.00                                                                             400.00                                                                             400.00                                                                             400.00                                                                             400.00                         KH.sub.2 PO.sub.4                                                                         --   --   --   --   60.00                                                                              --   --   --                             MgCl.sub.2.6H.sub.2 O                                                                     --   --   --   --   100.00                                                                             --   --   --                             MgSO.sub.4 (anhyd.)                                                                       --   --   97.67                                                                              --   --   --   --   --                             MgSO.sub.4.7H.sub.2 O                                                                     200.00                                                                             200.00                                                                             --   200.00                                                                             100.00                                                                             200.00                                                                             200.00                                                                             200.00                         NaCl        6460.00                                                                            5100.00                                                                            6460.00                                                                            6460.00                                                                            8000.00                                                                            6460.00                                                                            6460.00                                                                            6460.00                        NaHCO.sub.3 2200.00                                                                            2200.00                                                                            --   2200.00                                                                            350.00                                                                             2200.00                                                                            2200.00                                                                            2200.00                        NaH.sub.2 PO.sub.4.H.sub.2 O                                                              580.00                                                                             580.00                                                                             580.00                                                                             1400.00                                                                            --   580.00                                                                             580.00                                                                             580.00                         Na.sub.2 HPO.sub.4.7H.sub.2 O                                                             --   --   --   --   90.00                                                                              --   --   --                             OTHER COMPONENTS:                                                             Bacto-peptone                                                                             600.00                                                                             600.00                                                                             600.00                                                                             600.00                                                                             600.00                                                                             600.00                                                                             600.00                                                                             600.00                         Fetal Bovine Serum                                                                        --   --   --   --   --   c    c    c                              D-Glucose   3000.00                                                                            3000.00                                                                            3000.00                                                                            3000.00                                                                            1000.00                                                                            3000.00                                                                            3000.00                                                                            3000.00                        Glutathione (reduced)                                                                     0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50                           HEPES       --   5958.00                                                                            --   --   --   --   --   --                             Phenol red  10.00                                                                              10.00                                                                              10.00                                                                              10.00                                                                              10.00                                                                              10.00                                                                              10.00                                                                              10.00                          AMINO ACIDS:                                                                  L-Alanine   13.90                                                                              13.90                                                                              13.90                                                                              13.90                                                                              13.90                                                                              13.90                                                                              13.90                                                                              13.90                          L-Arginine.Cl                                                                             42.10                                                                              42.10                                                                              42.10                                                                              42.10                                                                              42.10                                                                              42.10                                                                              42.10                                                                              42.10                          L-Asparagine.sup.a                                                                        45.00                                                                              45.00                                                                              45.00                                                                              45.00                                                                              45.00                                                                              45.00                                                                              45.00                                                                              45.00                          L-Aspartic acid                                                                           19.97                                                                              19.97                                                                              19.97                                                                              19.97                                                                              19.97                                                                              19.97                                                                              19.97                                                                              19.97                          L-Cysteine.sup.b                                                                          31.50                                                                              31.50                                                                              31.50                                                                              31.50                                                                              31.50                                                                              31.50                                                                              31.50                                                                              31.50                          L-Glutamic acid                                                                           22.10                                                                              22.10                                                                              22.10                                                                              22.10                                                                              22.10                                                                              22.10                                                                              22.10                                                                              22.10                          L-Glutamine 219.20                                                                             219.20                                                                             219.20                                                                             219.20                                                                             219.20                                                                             219.20                                                                             219.20                                                                             219.20                         Glycine     7.50 7.50 7.50 7.50 7.50 7.50 7.50 7.50                           L-Histidine.HCl.H.sub.2 O                                                                 20.96                                                                              20.96                                                                              20.96                                                                              20.96                                                                              20.96                                                                              20.96                                                                              20.96                                                                              20.96                          L-Hydroxyproline                                                                          19.70                                                                              19.70                                                                              19.70                                                                              19.70                                                                              19.70                                                                              19.70                                                                              19.70                                                                              19.70                          L-Isoleucine                                                                              39.36                                                                              39.36                                                                              39.36                                                                              39.36                                                                              39.36                                                                              39.36                                                                              39.36                                                                              39.36                          L-Leucine   39.36                                                                              39.36                                                                              39.36                                                                              39.36                                                                              39.36                                                                              39.36                                                                              39.36                                                                              39.36                          L-Lycine.HCl                                                                              36.50                                                                              36.50                                                                              36.50                                                                              36.50                                                                              36.50                                                                              36.50                                                                              36.50                                                                              36.50                          L-Methionine                                                                              14.90                                                                              14.90                                                                              14.90                                                                              14.90                                                                              14.90                                                                              14.90                                                                              14.90                                                                              14.90                          L-Phenylalanine                                                                           16.50                                                                              16.50                                                                              16.50                                                                              16.50                                                                              16.50                                                                              16.50                                                                              16.50                                                                              16.50                          L-Proline   17.30                                                                              17.30                                                                              17.30                                                                              17.30                                                                              17.30                                                                              17.30                                                                              17.30                                                                              17.30                          L-Serine    26.30                                                                              26.30                                                                              26.30                                                                              26.30                                                                              26.30                                                                              26.30                                                                              26.30                                                                              26.30                          L-Threonine 17.90                                                                              17.90                                                                              17.90                                                                              17.90                                                                              17.90                                                                              17.90                                                                              17.90                                                                              17.90                          L-Tryptophan                                                                              3.10 3.10 3.10 3.10 3.10 3.10 3.10 3.10                           L-Tyrosine  18.10                                                                              18.10                                                                              --   18.10                                                                              18.10                                                                              18.10                                                                              18.10                                                                              18.10                          L-Tyrosine.2Na.2H.sub.2 O                                                                 --   --   26.10                                                                              --   --   --   --   --                             L-Valine    17.60                                                                              17.60                                                                              17.60                                                                              17.60                                                                              17.60                                                                              17.60                                                                              17.60                                                                              17.60                          VITAMINS:                                                                     Ascorbic acid                                                                             0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50                           Biotin      0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20                           Choline chloride                                                                          5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00                           D-Ca pantothenate                                                                         0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20                           Folic acid  10.00                                                                              10.00                                                                              10.00                                                                              10.00                                                                              10.00                                                                              10.00                                                                              10.00                                                                              10.00                          i-Inositol  36.00                                                                              36.00                                                                              36.00                                                                              36.00                                                                              36.00                                                                              36.00                                                                              36.00                                                                              36.00                          Niacinamide 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50                           Nicotinic acid                                                                            0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50                           Para-aminobenzoic acid                                                                    1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00                           Pyridoxal.HCl                                                                             0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50                           Pyridoxine.HCl                                                                            0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50                           Riboflavin  0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20                           Thiamine.HCl                                                                              0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20                           Vitamin B.sub.12                                                                          2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00                           __________________________________________________________________________    .sup.1 McCoy, T. A., Maxwell, M., and Kruse, P. F. (1959) Proc. Soc.          Exper. Biol. Med. 100, 115.                                                   .sup.2 Hsu, T. C. and Kellogg, D. S., Jr. (1960) J. Nat. Cancer Inst. 25,     221.                                                                          .sup.3 Iwakata, S. and Grace, J. T., Jr. (1964) N.Y.J. Med. 64:18, 2279.      .sup.4 McCoy's 5A Medium formulated with Hanks' and Suspension Salts is a     GIBCO modification and is not cited in                                        .sup. references 1-3.                                                         .sup.a HCl form listed by the Tissue Culture Standards Committee, In          Vitro (1974) 9:6.                                                             .sup.b Monohydrate form listed by the Tissue Culture Standards Committee,     In Vitro (1974) 9:6.                                                          c Fetal Bovine Serum Supplementation:                                         Cat. No.                                                                           FBS                                                                      320-6610                                                                           10% v/v                                                                  320-6620                                                                           20% v/v                                                                  320-6630                                                                           30% v/v                                                              

The serum component may be present in the culture in an amount of atleast 1% (v/v) to 50% (v/v). The serum concentration may be preferablyin the neighborhood of 15 to 30% (v/v). For higher serum concentrations,the exchange rate is increased proportionately. The third component maybe present in an amount of from 10⁻⁷ M to 10⁻⁴ M, and is preferablypresent in an amount of from 5×10⁻⁶ to 5×10⁻⁵ M. The media componentrepresents the balance such that all three components add up to 100%.Alternatively the serum component can be replaced by any of severalstandard serum replacement mixtures which typically include insulin,albumin, and lecithin or cholesterol. See, Migliaccio et al, Exp.Hematol. (1990) 18:1049-1055, Iscove et al, Exp. Cell Res. (1980)126:121-126, and Dainiak et al, J. Clin. Invest. (1985) 76:1237-1242.

Illustratively, human hematopoietic stem cell concentration may beincreased as follows. Red blood cells are removed from a bone marrowaspirate by ficol-hypaque gradient centriguration. The mononuclear cellsare then incubated with a "cocktail" of antibodies which recognizemature blood elements including red blood cells, granulocytes,macrophages, and mature lymphocytes (both B- and T-cells). In addition,antibodies are included which recognize committed progenitor cells(including anti CD 33). The mature cells are then removed by one ofvarious procedures including panning, magnetic beads, or cell sorting ona Fluorescent Activated Cell Sorter (FACS). By removing the matureelements, the chance of recombinant virus infection, and thereforetransfer of the desired gene(s), to hematopoietic stem cells is greatlyfacilitated.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

ILLUSTRATIVE CELL SEPARATION AND STAINING PROCEDURES Separation of BoneMarrow Cells on Ficoll

1. Dilute the bone marrow sample at a 1:4 ratio in I-MDM kept at roomtemperature (Iscove's Modified Dulbecco Medium; GIBCO; Cat. No.430-2200).

2. Carefully, layer 35 ml of the diluted bone marrow sample onto 15 mlof Ficoll-Paque at room temp. (Sp. Gr. 1.077 g/cc; Pharmacia; Cat. No.17-0840-02) in 50 ml centrifuge tube.

3. Centrifuge at 700 x g (1800 rpm on Beckman) for 30 min. at roomtemperature (20° C.).

4. After centrifugation remove most of the upper layer (leaving about 5ml above interphase), collect the interphase layer (bone marrow cells),and wash 3 times in ice cold I-MDM according to the following:

First wash: 1400 rpm/15 min/4° C.

Second wash: 1200 rpm/10 min/4° C.

Third wash: 1200 rpm/10 rpm/4° C.

5. After the third wash the cells are suspended either in media or abalanced salt solution etc. (depending on whtat is to be done withthem), and counted after making a 1:10 dilution of the cells in aceticacid (10 μl cell suspension=90 μl of 2% acetic acid in PBS; this willallow for counting WBCs only, since RBCs get lysed in the acetic acid).

6. Cells are then suspended to the desired final concentration in theappropriate medium (for media refer to the different applications).

Fluorescent Staining for MY-10 Positive Bone Marrow Cells

Reagents

Standard Buffer:

Powdered Bacto Dried DIFCO Buffer (Baxter; Cat. No. 2314-15GB): 200 g

10% NaN₃ (sodium azide): 20 g

Heat inactivated fetal calf serum (56° C., 30 min.) 200 ml

Bring volume to 20 liters in dd-H₂ O; pH 7.15-7.25; store at 4° C.

Good for 1 month

2% Paraformaldehyde solution

paraformaldehyde: 10 g

dd-H₂ O: 500 ml

10N NaOH (under the hood) 8-20 drops

powdered Bacto dried Difco Buffer 5g

Pour dd-H₂ O in 500 ml flask and stir on a hot plate to 60° C. in thehood.

Add 10 gr. parafromaldehyde

Add NaOH dropwise until solution clears

Add 5 gr DIFCO

Let cool, Adjust pH to 7.35-7.45 with 2N HCl.

1. After counting the cells, they are washed once in standard buffer(100 rpm, 5 min. 4° C.).

2. Cells are then suspended in standard buffer at the concentration of2×10⁵ cells/ml.

3. Two 50 μl aliquots of the cells are deposited into 2 15 ml centrifugetubes.

4. To one tube 50 μl of a 1:5 dilution of anti-HPCA-1 are added(anti-human progenitor cell antigen; Becton Dickinson; Cat. No. 7660;diluted 1:5 in standard buffer). To the other tube, 50 μl of a 1:5dilution of MIg are added (Mouse IgG1 control; Becton Dickinson; Cat.No. 9040; diluted 1:5 in standard buffer).

5. Both tubes are then incubated for 1/2 hour on ice.

6. After incubation; cells are washed twice in 5 ml standard buffer(1000 rpm, 5 min, 4° C.).

7. After the second wash, cell pellets are resuspended in 50 μl of a1:40 dilution of GAM-FITC (Affinity isolated Goat F(ab')2 anti-mouse IgGand IgM, human Ig adsorbed, Fluorescein conjugated; TAGO; Cat. No. 4353;diluted 1:40 in standard buffer).

8. Cells are incubated for 1/2 and hour in the dark, on ice.

9. After incubation, cells are washed twice in 5 ml standard buffer (100rpm, 5 min , 4° C.) and each of the pellets is resuspended in 100μstandard buffer plus 100 μl 2% paraformaldehyde solution.

10. Cells are then analyzed for fluorescence using the flow cytometer.Percent positive fluorescence constitute % fluorescence in anti-HPCA-1sample minus % fluorescence in MIg sample.

Fluorescent Staining of Bone Marrow Cells to Sort Out Mature ProgenitorCells

Objectives:

The purpose of this staining is to enrich for hematopoietic stem cells(most primitve stem cells) by removing mature cell populations, usingthe flow cytometer or magnetic beads. It is always a good idea to retainsome cells as total bone marrow cells (after Ficoll separation) to stainfor MY-10 positive cells in order to compare with the outcome of sortingand determine extent of enrichment.

1. Cells are separated on Ficoll-Paque as described before. (Remove0.5×10⁶ cells and divide into 2 portions to stain withanti-HPCA-1/GAM-FITC and MIg/GAM-FITC).

2. After the third wash the cell pellet is suspended in the monoclonalantibody cocktail (refer to section describing the making of thiscocktail) using 1 ml of the cocktail per 10⁷ cells, and cells areincubated on ice for 1 hour.

3. Cells are then washed three times in excess ice cold I-MDM (1000 rpm,5 min, 4° C.).

4. After the third wash cells are suspended in a 1:40 dilution ofGAM-FITC (diluted in I-MDM, not standard buffer) at the rate of 50 μlper 0.25×10⁶ cells, and incubated on ice in the dark for 1/2 an hour.

5. After incubation, cells are washed three times in ice cold I-MDM, andafter the final wash they are suspended in 2-4 ml of ice cold I-MDM andkept on ice until sorting.

6. Cells are then sorted on the flow cytometer based on fluorescence, toeclude the upper 85% of the fluorescence histogram. Sorting could berepeated twice for better enrichment.

7. After sorting, cells are counted, washed, and an aliquot is stainedfor MY-10 positive cells (as described above) to determine the extent ofenrichment in comparison to stained aliquots of the total bone marrowcells.

Selecting for Immature Cells by Using Magnetic Antibodies

1. Follow steps 1-3 in the procedure for Fluorescent staining of bonemarrow cells to sort out mature cells. Note: sodium azide is notincluded in any of the buffers.

2. In the meantime wash an appropriate amount of magnetic goatanti-mouse Ig (Biomag; Collaborative Research; Cat. No. 74340-50; 1mg/ml; 5×10⁸ particles/ml) 3 times in ice cold I-MDM at 1500 rpm, 5 min.4° C. (to wash off the sodium azide which is used as a preservative).

3. Resuspend the cell pellet obtained after the third wash in "step 1"in Biomag at the rate of 50 particles Biomag/cell (e.g., for 1×10⁶ cellsuse 5×10⁷ particles, therefore, 0.1 ml of Biomag).

4. Deposit the cells in a T-25 or T-75 tissue culture flask (dependingon cell numbers) and incubate on ice for 1/2 hour with intermittentshaking.

5. After incubation, lay the flask onto the flat magnet (provided withthe Biomag), secure with a rubber band or tape, and incubate at 4° C.for 10-15 min.

6. Stand the magnet and the flask into an upright position and collectthe supernatent.

7. Repeat steps 4-6 two more times.

8. Count the cells, wash once in ice cold I-MDM, remove an aliquot tostain for MY-10 positive, and resuspend in appropriate media for furtheruse.

I. Medium Replacement

Materials and Methods:

Cells: Human bone marrow cells were obtained from heparinized aspiratesfrom the iliac crest of informed and consenting individuals. The bonemarrow was separated by a Ficoll-Paque (Pharmacia, No. 17-0840-02)density gradient centrifugation and the low density cells (<1.077gm/cm³) were collected and washed 3 times with Iscove's ModifiedDulbecco's Medium (IMDM). The cells were counted between the second andthird washes. The cells were then seeded onto 24-well tissue cultureplates (Costar No. 3524) in duplicate or triplicate at 1, 2, and 5·10⁶cells/ml at 322 μl/well.

Long-term culture conditions: The low density cells were incubated inIMDM supplemented with 10% fetal calf serum (Hyclone Laboratories, 10%horse serum (Hyclone Laboratories), 1% penicillin/streptomycin (Sigma,10,000 U/ml penicillin G and 10 mg/ml streptomycin, Cat. No. P3539), and10⁻⁵ M hydrocortisone (17-Hydroxy-corticosterone, Sigma, Cat. No. H0888)in a humidified 5% CO₂ /95% air atmosphere. The cultures were treatedwith one of three medium exchange schedules, 100% daily medium exchange(7/wk), 50% daily medium exchange (3.5/wk), or 50% biweekly mediumexchange (1/wk). Twice per week during the medium exchange, 50% of thenon-adherent cells were removed from each culture well and counted usinga hemocytometer.

When the cells were removed for counting (twice/week), all of the mediumremoved during feeding of the 3.5/wk and 1/wk cultures was saved forcell counts and fresh medium returned to the wells. The 7/wk culturesrequired saving 1/2 of the removed medium for cell counts, whilecentrifuging and returning the non-adherent cells in the remaining 1/2of the medium removed. Fresh medium was then added to each well toreplace the medium removed for cell counts. On days when the cells werenot removed for counting, 100% or 50% of the medium was removed fromeach of the 7/wk and 3.5/wk culture wells respectively, the cells werecentrifuged and returned to the original wells with additional freshmedium.

Methylcellulose and morphologic assays: One every other week thenon-adherent cells removed for cell counts were plated inmethylcellulose in the presence of erythropoietin, GM-CSF, and IL-3, andthe Granulocyte Macrophase-Colony Forming Units (CFU-GM) wereenumerated. Aliquot of removed cells were cytocentrifuged, stained withWright-Giemsa, and differential cell counts performed.

Statistical analysis: The biweekly cell production results are expressedas the mean ±SEM from replicate cultures. The probability of significantdifferences between groups of cultures was determined by comparing thenormalized cumulative cell production values from the rapidly exchangedcultures (7/wk and 3.5/wk) to the matched control cultures (1/wk) usinga paired t-test. Statistical significance was taken at the 5% level.

Results:

Kinetics of nonadherent cell production: Nonadherent cell production wasexamined both as a function of inoculum cell density (over the range1-5·10⁶ cells/ml) and medium exchange rate. The medium exchange rate wasvaried from one medium volume exchange per week, the traditional Dexterculture rate, to seven medium volume exchanges per week. The biweeklynumber of cells collected was normalized by dividing by the number ofcell inoculated per culture.

At each medium exchange rate, the normalized cell collection curves didnot change significantly with inoculum density. The cell production forthe cultures maintained at the three medium perfusion rates of 7/wk,3.5/wk and 1/wk were similar when normalized to the number of cellsinoculated per culture. Comparison of the final cumulative cellproductions between inoculum densities showed no significantdifferences, at any of the three medium exchange rates (p>0.20 by apaired t-test for all pairs of samples).

The medium exchange rate, in contrast, strongly influenced the rate andlongevity of cell production in these cultures. Cell production of thecultures exchanged at 1/wk (control), 3.5/wk, and 7/wk all decayed overthe first few weeks. Differences in culture productivity, however,became apparent after week 3 in culture. Between weeks 3 to 10, the cellproduction was constant in the 7/wk cultures, constant at a lower levelin the 1/wk cultures, but increased exponentially in the 3.5/wkcultures. After weeks 10 to 12, cell production declined in all culturesuntil culture termination.

Results for the 1/wk exchanged cultures are equivalent to those commonlyobserved in traditional human Dexter cultures in a variety of systems,whereas the rapidly exchanged cultures of 3.5 and 7/wk showed increasedcell productivity when compared to previous optimum culture methods.Cultures in which 1/2 of the medium was exchanged daily (3.5/wk)maintained increased cell production for substantially longed thaneither the control (1/wk) or complete daily exchange (7/wk) cultures.Between weeks 3 and 9, the number of nonadherent cells collected fromthe 3.5/wk exchanged cultures increased exponentially with a doublingevery 2.1 weeks.

The cell production under the 3.5/wk and 1/wk protocols can be directlycompared by plotting the cell production under the 3.5/wk exchange rateas a percentage of the production of the cultures with an exchange rateof 1/wk. This comparison shows that during the initial decay phase thecell production under the two protocols is similar. However, betweenweeks 3.5 and 18, the cell production under the 3.5/wk exchange rate isconsistently higher.

The proliferative potential of the cultures can thus be measured bytheir ability to produce cells following the initial decay. Thenormalized cumulative cell production following week 3 (Σ_(i=7) ^(n),C_(i) /C_(o) was independent of the cell inoculation density for themedium exchange rates of 7/wk, 3.5/wk. Cell production data from thecultures at similar medium exchange rates were qualitatively andstatistically similar, and were therefore density averaged and combined(bottom panel) to obtain a larger statistical sample. The densityaveraged cumulative cell production between weeks 3.5 and 20 was: 0.22for the 7/wk; 0.40 for the 3.5/wk; and 0.15 for the 1/wk cultures. Theincrease in the medium exchange rate from 1/wk to 7/wk thus increasedthe cell production about 60% over the typical Dexter culture mediumexchange schedule. The 3.5/wk exchange rate resulted in almost 3-foldcumulative cell production increase compared to the 1/wk Dexterprotocol. Statistical analysis of these data using a paired t-test,demonstrated significant differences between both the 7/wk vs. 1/wk andthe 3.5/wk vs. 1/wk at the 5% level of significance. The medium exchangerate of 3.5/wk thus improves the cell production rate over thetraditional Dexter protocol of 1/wk.

Granulocyte-Macrophase Progenitor Cell Production

Granulocyte-macrophase progenitor cell assays were performed fromreplicates of a given medium perfusion schedule and inoculum density(Table 2). The medium perfusion rate had a pronounced effect on thenumber of granulocyte-macrophage progenitor cells produced. The 3.5/wkmedium exchange cultures showed the greatest longevity in terms ofprogenitor cell production. These cultures produced progenitors at astable rate between weeks 4 and 18.

The optimum conditions in terms of progenitor cell production are thecultures exchanged 3.5 times per week and inoculated at 5·10⁶ cells/ml.These cultures produced a significant number of progenitor cells untilweek 20. Statistical analysis, using a paired t-test, showed that theoptimum medium exchange rate cultures of 3.5/wk produced significantlymore granulocyte-macrophage progenitor cells after week 8 than did thecorresponding 7/wk and 1/wk cultures at all three inoculation densitiesat the 1% level of significance. The number of progenitor cells producedis important as it is an indirect measure of stem cell renewal.Progenitor cells can only be present after several weeks in culture bydifferentiation from an earlier cell, presumably a stem cell, which isstill present in culture. Thus, these data suggest that morephysiologic, rapid medium/serum exchange rate and higher cell densitiesmay have provided conditions that supported some degree of stem cellrenewal for five months.

Nonadherent cell morphology: To determine whether the prolongedhematopoiesis supported by the 3.5/wk cultures was qualitativelydifferent from the other cultures, the non-adherent cells collectedbetween weeks 10 and 19 were stained and typed morphologically. At theexchange rates of 1/wk and 7/wk, the cells produced were mostlymacrophages by week 15 and thereafter (Table 3), which is similar toresults from studies in other laboratories. In contrast, the culturesperfused at a rate of 3.5 medium volumes per week and seeded at 5·10⁶cells/ml produced granulocytes as well as macrophages through week 19.Thus, it seems that this medium exchange rate and inoculum density moreeffectively reconstituted granulopoiesis in vitro.

                                      TABLE 2                                     __________________________________________________________________________    The average number of nonadherent progenitor cells removed from long term     bone marrow                                                                   cultures (LTBMCs as a function of the medium perfusion rate and inoculum      density.                                                                               7/wk             3.5/wk           1/wk                                   5 × 10.sup.6                                                                 2 × 10.sup.6                                                                  1 × 10.sup.6                                                                 5 × 10.sup.6                                                                  2 × 10.sup.6                                                                  1 × 10.sup.6                                                                 5 × 10.sup.6                                                                  2 × 10.sup.6                                                                  1 × 10.sup.6           Week                                                                              per ml                                                                             per ml                                                                              per ml                                                                             per ml                                                                              per ml                                                                              per ml                                                                             per ml                                                                              per ml                                                                              per ml                       __________________________________________________________________________     2  237 ± 27                                                                        11 ± 3.3                                                                         106 ± 5                                                                         120 ± 16                                                                         132 ± 7.9                                                                        167 ± 13                                                                        368 ± 29                                                                           94 ± 20.8                                                                      335 ± 46                   4  149 ± 21                                                                        101 ± 5.1                                                                        104 ± 10                                                                         93 ± 10                                                                          37 ± 5.6                                                                        20 ± 0                                                                          21 ± 1.3                                                                         2 ± 0                                                                              8 ± 4.4                  6  47.7 ± 7                                                                        12 ± 2.5                                                                         8 ± 0                                                                           17 ± 3                                                                            6 ± 4.1                                                                           5 ± 2.7                                                                       13 ± 5.1                                                                         1 ± 0                                                                             1 ± 0                     8  40 ± 3                                                                          0     4 ± 0                                                                           38 ± 6                                                                            24 ± 2.7                                                                        10 ± 3                                                                          34 ± 7.4                                                                         0     0                            10  0    0     0      28 ± 8.3                                                                        10 ± 2.9                                                                          5 ± 1.3                                                                        8 ± 2.3                                                                           2 ± 2.3                                                                        0                              12.5                                                                            0     6 ± 2.3                                                                         0      8 ± 2.3                                                                        0     0    0     0     0                            14  0    0     0      22 ± 6.4                                                                        6 ± 1.3                                                                          2.5 ± 1.2                                                                       3 ± 1.3                                                                         0     0                            16    6 ± 2.2                                                                       0     0      24 ± 7.6                                                                        4 ± 1.7                                                                           2 ± 1.3                                                                        9 ± 3.6                                                                         0     0                            18  0    0     0      24 ± 6.3                                                                        4 ± 1.3                                                                         0    0     0     0                            20  0    0     0     5 ± 0                                                                            4 ± 0                                                                            3 ± 0                                                                          1 ± 0                                                                            0     0                            22    2 ± 1.3                                                                       0     0      4 ± 1.3                                                                        10 ± 3                                                                           0    0     0     0                            10-22*                                                                              8 ± 3.5                                                                        6 ± 2.3                                                                         0      115 ± 32.2                                                                      40 ± 11.2                                                                       12.5 ± 3.8                                                                       21 ± 7.2                                                                        2 ± 7                                                                            0                            __________________________________________________________________________     Replicate samples at each medium perfusion rate and inoculum density were     pooled and are each tabulated as one mean ± SEM. Cumulative CFUGM          production after week 8 is statistically greater in the 3.5/wk cultures       than the corresponding cultures perfused at 7/wk or 1/wk at all inoculum      densities at the 1% level of significance.                               

This result supports the hypothesis that long-term human Dexter cultureconditions are suboptimal and as a culture in vitro better approximatethe hematopoietic environment in vivo, more effective reconstitution ofbone marrow ex vivo can be attained.

Physical appearance: The medium exchange rate significantly affected thephysical appearance of the cultures. By 10 weeks in culture, the 7/wkcultures had large number of adipose cells in the stroma while the3.5/wk cultures had few fat cells and the 1/wk cultures never developedfat cells. At culture termination at 26 weeks, the stroma of the 7/wkcultures were composed of approximately 20-30% fat cells while the3.5/wk cultures still only had a few fat cells. Adherent colonydistribution also varied between cultures with different mediumperfusion rate. Adherent colonies in the 3.5/wk cultures persistedlonger than those in the 7/wk and 1/wk cultures.

                                      TABLE 3                                     __________________________________________________________________________    Nonadherent cell morphology as a function of the                              medium perfusion rate and inoculum density.                                            5 × 10.sup.6 per ml                                                                2 × 10.sup.6 per ml                                                                1 × 10.sup.6 per ml                                      %          %          %                                       Medium          myeloid    myeloid    myeloid                                 perfusion       pre-       pre-       pre-                                    rate weeks                                                                             % M.o slashed.                                                                    % G                                                                              cursors                                                                           % M.o slashed.                                                                    % G                                                                              cursors                                                                           % M.o slashed.                                                                    % G                                                                              cursors                                 __________________________________________________________________________    7/wk 10.4                                                                              25  57 18  57  32 11  52  34 14                                           13.4                                                                              49  34 17  92   5  3  63  22 15                                           15.4                                                                              66  19 16  79  19  2  54  17 29                                           19  93   5  1  96   3  1  100  0  0                                      3.5/wk                                                                             10.4                                                                              50  27 23  45  38 17  39  45 17                                           13.4                                                                              23  59 19  27  56 17  36  47 17                                           15.4                                                                              41  38 21  44  27 29  67  13 21                                           19  58  37  5  88   9  3  99   1  0                                      1/wk 10.4                                                                              59  21 20  60  11 29  ND  ND ND                                           13.4                                                                              56  25 20  19  36 46  43   7 50                                           15.4                                                                              76   4 20  ND  ND ND  46  39 15                                           19  100  0  0  100  0  0  100  0  0                                      __________________________________________________________________________     Data are for pooled replicate samples at each medium perfusion rate and       inoculum density and are shown as the percentage of macrophages (% M.o        slashed.), granulocytes (mature granulocytes and bands, % G), and immatur     granulocytes (metamyelocytes and less mature cells, % myeloid precursors)                                                                              

II. Medium Replacement Combined with Supplementation of Medium withHematopoietic Growth Factors

Materials and Methods:

Cells: Human bone marrow cells were obtained following informed consentfrom heparinized aspirates of the iliac crest bone marrow, under aprotocol approved by the University of Michigan Human InvestigationCommittee. The bone marrow was separated by a Ficoll-Paque (Pharmacia)density gradient centrifugation and the low density cells (<1.077gm/cm³)were collected and washed 3 times with IMDM. The cells were countedbetween the second and third washes. The cells were then seeded onto6-well tissue culture plates (Costar No. 3406) or collagen coated 6-wellplates (rat tail type 1 collagen, Biocoat. Collaborative Research Inc.Cat. No. 40400) in duplicate 5.106 cells/ml at 1.5 ml/well.

Culture medium: The medium used was IMDM (Gibco Laboratories. Cat. No.430-2200) containing 10% fetal calf serum (Hyclone Laboratories), 10%horse serum (Hyclone Laboratories), 1% penicillin/streptomycin (Sigma,10,000 U/ml penicillin G and 10 mg/ml streptomycin, Cat. No. P3539), and10⁻⁵ M hydrocortisone (17-Hydroxycorticosterone, Sigma, Cat. No. H0888).

Hematopoietic growth factors (HGH): Due to the frequent culturesupplementation via rapid medium exchange, hematopoietic growth factorswere added to the medium at approximately 1/20 of the concentrationsfound to promote maximal colony formation in clonal assays 4. Theconcentrations used were 1 ng/ml of IL-3, 1 ng/ml of GM-CSF (AmgenBiologicals, Cat. No. 13050), 0.1 U/ml of Epo (Terry Fox Labs.Vancouver, Canada).

Hematopoietic progenitor cell assay: Nonadherent hematopoietic cellsremoved from culture were counted and plated at 1·10⁵ cells/ml or fewercells in methylcellulose. GM-CSF and Epo were added to themethylcellulose at 20 ng/ml and 2 U/ml, respectively. The cell wereplated in 24 well plates at 0.25 ml/well and incubated at 37° C. for 14days. The colonies were then counted under an inverted microscope andcolonies greater than 50 cells were scored as GM-colony forming units(CFU-GM), erythroid burst-forming unit (BFU-E), or granulocyte erythroidmegakaryocyte macrophage-colony forming unit (CFU-GEMM).

LTBMC conditions: The cultures were incubated at 37° C. in a humidified5% CO₂ /95% air atmosphere and perfused (medium exchanged) at a rate of50% daily medium exchange. During the first week in culture, all cellsremoved during the daily medium exchange were centrifuged and returnedto the original wells. After the first week in culture, 50% of the totalnonadherent cells were removed from the cultures on a biweekly basisduring the medium exchange, mononucleated cells counted, and freshmedium returned to the wells. The remaining five days per week when thecells were not counted, 50% of the medium was removed from each of theculture wells and replaced with fresh medium, the removed medium wascentrifuged, the medium decanted from the cell pellet, and the cellsreturned to their original wells.

Statistical analysis: The probability of significant differences betweengroups of cultures was determined by comparing the normalized cumulativecell production values from the rapidly perfused cultures supplementedwith hematopoietic growth factors to the matched untreated controlcultures using a paired t-test. Statistical significance was taken atthe 5% level. There were no statistical differences between matchedrapidly perfused LTBMCs cultured on tissue culture plastic and type Irat tail collagen at the 5% level. Therefore, the data for the plasticand collagen matrix were combined for presentation in this and all otherfigures and statistical analysis performed on the combined data.

Results:

Kinetics of cell production in rapidly exchanged growth factorsupplemented LTBMCs: As a first test of the hypothesis that thelongevity and productivity of long term bone marrow cultures (LTBMCs) islimited by insufficient production of HGF's, we maintained rapidlyexchanged ex vivo bone marrow cultures that were supplemented with IL-3,or Epo. In these cultures, 50% of the medium was removed daily andreplaced with an equal volume of fresh medium supplemented with IL-3 orEpo. The cells removed were then centrifuged, the medium decanted anddiscarded, the cells resuspended, and the cells returned to the originalcultures. IL-3 and Epo individually enhanced the cell productivity ofrapidly exchanged LTBMCs. The cultures containing Epo alone initiallyhad a high cell production rate due to substantial terminal erythroiddifferentiation. However, by week four erythropoiesis had ceased and thecell production rate had decreased to the level of the control cultures.IL-3 and Epo induced an average increase in nonadherent cell productionover controls throughout the 18 weeks of culture of 175% and 173%,respectively.

Combinations of growth factors proved to be more effective in increasingthe nonadherent cell production rate. The highest rate of cellproduction was observed for the combination of IL-3+GM-CSF+Epo. Thesecultures produced approximately 25% of the number of cells inoculatedbiweekly during the first 6 weeks in culture and had an average 4.8-foldincrease in nonadherent cell production over controls during weeks 2-8.The combination of IL-3+GM-CSF produced an average 3.5-fold increase innonadherent cells as compared to controls through week 8. In separateexperiments, adding neither IL-6 nor G-CSF to the combination ofIL-3+GM-CSF+Epo improved the nonadherent cell production rate, butinstead resulted in cell production rates indistinguishable from thecultures containing the combination of IL-3+GM-CSF. In all cases, thestimulatory effect on cell production induced by the addition of HGFswas maximal between weeks 0 to 8, although cell production was higherthan the controls throughout the culture.

The combinations of HGFs lead to high absolute numbers of nonadherentcells produced in rapidly exchanged LTBMCS. The productivity of thecultures can be shown by comparing the cumulative number of cellsproduced over time (Σ_(i=1) ^(n), C_(i), C_(i) being the number ofnonadherent cells collected at time i), relative to the number of cellsinoculated (C_(o)) by plotting the ratio (Σ_(i=1) ^(n), C_(i), C_(o)) asa function of time. When this ratio exceeds unity, a culture hasproduced more cells than were inoculated and the culture has led to anexpansion in cell number.

The combination of IL-3+GM-CSF+Epo induced cumulative cell productionthat was more than 3-fold greater than the number of cells inoculated.The cell production rate was the highest during the first 6 weeks inculture during which time the culture produced approximately as manycells as were inoculated every two weeks. This maximum cell productionrate was 15% of the estimated in vivo bone marrow cell production ratewhere 50% of the myeloid cell mass is generated daily. The combinationof IL-3+GM-CSF resulted in more than a 2-fold expansion in cell numberand at rates comparable to the combination of IL3+GM-CSF+Epo duringweeks 3-7 in culture. Untreated rapidly exchanged (50% daily mediumexchange) and slowly exchanged (50% medium exchange biweekly) controlcultures not supplemented with HGFs produced approximately 1 and 0.37times the number of cells inoculated after 18 weeks, respectively. Moreimportantly more than half of all cells removed from theseunsupplemented cultures came from the first two samplings, indicatingthat many of these cells were from the original inoculum and thatsupplementation of the cultures with HGFs are required to inducesignificant cycling of progenitor and stem cells.

Morphologic analysis of nonadherent cells: The addition of multiple HGFsalso increased the variety of myeloid cells produced in the cultures.The control cultures produced nonadherent cells that were predominatelymacrophages after week 3 in the culture. Production of erythroid cellsdecreased rapidly with few erythroid cells detected after week 5. Thecultures containing Epo (Epo alone, IL-3+Epo, and IL-3+GM-CSF+Epo)produced a transient increase in erythroid cell production, with a highpercentage (55-75%) of nonadherent cells being erythroid through week 3.When IL-3+Epo±GM-CSF was present, the cultures continued to produceerythroid cells throughout the 16 weeks in culture with about 5-15% ofthe nonadherent cells being typed as erythroid. Thus, in the presence ofIL-3+Epo, erythropoiesis was active throughout.

IL-3±Epo led to a nonadherent cell population that was predominately(60-70%) late granulocytes (LG) at week 5. The percentage of LGssteadily declined until it reached about 20% at week 18. The productionof macrophages rose correspondingly. When GM-CSF was added to IL-3±Epo,the high percentage of LG persisted through 18 weeks. The combination ofIL-3+GM-CSF thus led to active granulopoiesis for 18 weeks in culture,and the addition of Epo maintained erythropoiesis as well.Photomicrographs of the control and IL-3+GM-CSF+Epo supplementedcultures at 5.5 weeks in culture show the dramatic enhancement inculture density and variety of cells produced.

Kinetics of nonadherent progenitor cell production: Progenitor cellproduction increased with the addition of multiple HGFs. The productionof granulocyte macrophage colony forming units (CFU-GMs) in theuntreated controls was prolonged and steady for over 18 weeks, which isconsistent with the earlier results obtained using rapidly perfusedLTBMC without HGF. CFU-GM produced in the IL-3+GM-CSF andIL-3+Epo±GM-CSF cultures was approximately 10-fold higher than controlsduring weeks 3 to 5.

Erythroid burst forming unit (BFU-E) production in human LTBMC has beenreported to be low and cease quickly (Coutinho et al, Blood (1990)75(11): 2118-2129). The rapidly exchanged, untreated controls exhibiteda rapid decrease in BFU-E production although low levels of BFU-E wereproduced through 17 weeks in culture. The addition of Epo alone did notsignificantly influence the number of BFU-Es produced. IL-3 aloneinduced a mild short-lived stimulation of BFU-E production in weeks 3-5.On the other hand, IL-3 plus either Epo or GM-CSF induced a 10 to20-fold elevation of nonadherent BFU-E levels compared to that ofcontrols during weeks 3 to 5 of culture.

III. Transformation of Human Stem Cells

Materials and Methods

Cells

Human bone marrow cells were obtained following informed consent fromheparinized aspirates of the iliac crest bone marrow, under a protocolapproved by the University of Michigan Human Investigation Committee.The bone marrow was separated by a Ficoll-Paque (Pharmacia) densitygradient centrifugation and the low density cells (<1.077 gm/rm³) werecollected and washed 3 times with IMDM. The cells were counted betweenthe second and third washes. For the CD18 gene transfer experiments,bone marrow was obtained following informed consent from a CD18deficient patient donor.

Lineage negative (Lin⁻) selection of bone marrow cells

Mature mononuclear cells were removed from the above cell preparation byincubating the cells with a mixture of monoclonal antibodies (MAb) afterthe third wash with IMDM. 10⁷ cells were incubated in 1 ml of MAbcocktail on ice for 1 hour with gentle mixing every 10-15 minutes. TheMAb cocktail used is shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Preparation of monoclonal antibodies used to                                  separate lineage.sup.+ bone marrow mononuclear cells.                                                          Conc.                                        Monoclonal                       μg/10.sup.9                                                                      Volume                                 Antibody   Specificity  Cat. No. cells (μl)                                ______________________________________                                        Anti--Leu-1                                                                              T-cell       6300     30    125                                    Anti--Leu-5b                                                                             T-cell, E-rosette                                                                          7590     32     16                                               receptor                                                           Anti--Leu-10                                                                             B-cells, monocytes                                                                         7450     250   125                                    (Anti--HLA--DQ)                                                               Anti--Leu-12                                                                             B-cells      7540     120    60                                    Anti--CALLA                                                                              Common Acute 7500     60     60                                               Lymphoblastic                                                                 Leukemia                                                           Anti--Leu-M1                                                                             Monocytes,   7420     1000  500                                               granulocytes                                                       Anti--MO-1 Macrophages  --       1000 μl                                                                          1000                                   Anti-10F-7 RBCs         --       2000 μl                                                                          2000                                   Anti-E3    RBCs         --       2000 μl                                                                          2000                                   ______________________________________                                         Add IMDM to make MAb cocktail total volume of 100 ml, filter sterilize,       aliquot in 1 ml columns, and store at -20° C.                     

The cells were washed 3 times in excess ice cold IMDM and centrifuged at4° C. An appropriate amount of magnetic goat anti-mouse Ig (Biomag;Collaborative Research Corp., Cat. No. 74340-50, 1mg/ml, 5×10⁸particles/ml) was washed 3 times in ice Cold IMDM and centrifuged. Thecells were resuspended in Biomeg at 50 particles/cell and placed in aT-25 or T-75 tissue culture flask and incubated on ice for 1/2 hour withintermittent shaking. After incubation, the flask was laid onto flatmagnet, the magnet secured to the flask and incubated at 4° C. for 10-15minutes. The magnet and flask were stood upright and the supernatantcollected. The incubation with shading, adding magnet, standing upright,and collecting supernatant was repeated 2 more times. The cells werecounted and seeded onto 6-well tissue culture plates. (Costar No. 3406).

Culture medium

The medium used was IMDM (Gibco Laboratories, Cat. No. 430-2200)containing 10% fetal calf serum (Hyclone Laboratories), 10% horse serum(Hyclone Laboratories), 1% penicillin/streptomycin (Sigma, 10,000 U/mlpenicillin G and 10 mg/ml streptomycin, Cat. No. P3539), and 10⁻⁵ Mhydrocortisone (17-Hydroxycorticosterone, Sigma, Cat. No. H0888).

Hematopoietic growth factors

The hematopoietic growth factors were the optimum discussed above. Theconcentrations used were 1 ng/ml or 0.4 U/ml of IL-3 (a gift fromGenetics Institute, Cambridge, Mass.), 1 ng/ml of GM-CSF (a gift fromGenetics Institute, Cambridge, Mass.), 50 U/ml of IL-1a (Genzyme Corp.),0.1 U/ml of Epo (Terry Fox Labs, Vancouver Canada), 10 ng/ml MGF (mastcell growth factor, the c-kit ligand, Immunex Corp., Seattle, Wash.),and 2.0 ng/ml Hybrikine ( PIXY321! Immunex Corp., Seattle, Wash.).

Hematopoietic progenitor cell assay

Nonadherent hematopoietic cells removed from culture during the weeklysampling were counted and plated at 1-10⁵ cells/ml or fewer cells inmethylcellulose. MGF, GM-CSF and Epo were added to the methylcelluloseat 50 ng/ml, 20 ng/ml, and 2 U/ml, respectively. The cells were platedin 24 well plates at 0.25 ml/well and incubated at 37° C. for 14 days.The colonies were then counted under an inverted microscope and coloniesgreater than 50 cells were scored as a GM-colony forming units (CFU-GM),erythroid burst-forming unit (BFU-E), or granulocyte erythroidmegakaryocyte macrophage-colony forming unit (CFU-GEMM).

Retroviral producer cell lines

Two retroviral producer cell lines were obtained from Dr. Eli Gilboa'sLaboratory at Memorial Sloan Kettering Cancer Center, New York, N.Y. Thecell line produce amphotrophic viral particles that contain the NEO genewhich produces neomycin phosphotransferase providing resistance to themammalian neomycin analog G418. Also both cell lines produce retroviralparticles that are deficient in the required retroviral genes so thatcells infected with the retrovirus cannot themselves produce infectiousvirus.

The SAX containing packaging cell line is a 3T3 based cell line whichcontains a modified Moloney Murine Leukemia Virus (MoMuLV). The SAXprovirus contains the NEO gene, an SV40 promoted Adenosine deaminasegene, in a Xhol restriction site. Also, the SAX provirus contains the ψpackaging region but is deficient in the gag (core proteins), pol(reverse transcriptase), and env (envelope proteins) genes. This secondretroviral particle contains a double copy of the foreign DNA and theretroviral particles are denoted DC-29 (double copy-29th clone). TheDC-29 provirus contains two copies of the NEO gene and other retroviraland foreign DNA in a 3T3 cell line.

For the CD18-experiments, the amphotropic packaging cell Psi-Cripinfected with a retroviral vector containing a human full length CD19cDNA was used (Wilson et al, Science (1990) 248:1413-1416). In thisretrovirus, a full length cDNA for human CD18 was cloned into the BamH1site of a vector that expresses the recombinant gene from heterologoussequences spanning the 5' region of the chicken β-actin gene calledBA-CD18. Sequences 5' to the immediate early (IE) gene of humancytomegalovirus were subcloned into PUC19 and a portion containing IEenhancer sequences was removed on a Xho 1(from the polylinker) to Nco1(-220 of the IE gene) fragment). Synthetic linkers were used to convertthe Ncol site to a Xhol site and the modified fragment was cloned intothe unique Xhol site of BA-CD18 located 5' to the β-actin promoter. Thisnew vector was called CMV-BA-CD18.

Retroviral particle production

The SAX retroviral particles were provided by Dr. Clay Smith in Dr. EliGilboa's laboratory as viral supernatant solutions frozen and stored at-80° C. The DC-29 and CD18 retroviral particles were produced by growingthe DC-29 and CD18 viral packaging line to near confluency in a T-75flask, changing all the medium, incubating the cells for 12-15 hours andthen collecting the medium containing the viral particles. The viruscontaining supernatant was then centrifuged to remove vital packagingcells, the medium removed, and frozen in aliquots at -80° C.

LTBMC with supernatant added SAX retrovirus. DC29 retrovirus, or CD18retrovirus

The cultures were incubated at 37° C. in a humidified 5% CO₂ /95% airatmosphere. During the two first weeks in culture, two thirds of themedium (1 ml) was removed from each culture well daily and the mediumreplaced with an equivalent volume fresh medium containing HGF's (0.85ml) and viral cell producer supernatant; (0.15 ml). The retroviralsupernatant containing medium was thawed immediately prior to use and ifnot completely urged, it was stored on ice in a refrigerator. The mediumremoved from the cultures was centrifuged, the medium decanted, and thecells returned to the original wells.

LTBMC co-cultured with SAX retrovirus packaging cell line

The SAX retrovirus packaging cell line was grown to approximately 10%confluency in T-25 flasks (Costar, No. 3056) and then subjected to 2000rads of radiation. The hematopoietic cells prepared above were added tothe irradiated viral producer cells and cultured wit 50% daily mediumexchange for 2.5 weeks with all cells being returned to the wells. At2.5 weeks in culture, a 0.5 mM solution of EDTA was added to the flasksto remove the hematopoietic cells while leaving the stroma. The removedhematopoietic cells were added to 3 wells of a 6 well plate with 1000freshly trypsinized bone marrow fibroblast cells per well.

Sampling of infected LTBMCs

Beginning at week two in culture, after retrovirus addition had ended orco-culture had ceased, the cultures had 50% medium exchanged per daywith the nonadherent cells in the exchanged medium being removed onceper week for analysis. Nonadherent cells were removed from the culturesduring the daily medium exchange, the mononucleated cells counted, andfresh medium returned to the wells. The remaining six days per week whenthe cells were not counted, 50% of the medium was removed from each ofthe culture well sand replaced with fresh medium, the removed wascentrifuged, the medium decanted from the cell pellet, and the cellsreturned to their original wells.

Analysis for retroviral infection

The initial bone marrow inoculum was plated in 0, 0.4, 0.8, 1.2, 1.6,and 2.0 mg/ml G418 to obtain a kill curve to determine the concentrationof G418 in which to plate the post-infected bone cells. Cells removedfrom the cultures were plated in methylcellulose with G418 at 0.0, 0.8,and 1.6 mg/ml G418. After two weeks, the number of progenitor cellcolonies were enumerated in the methylcellulose. Individual colonieswere then plucked from the methylcellulose and assayed by polymerasechain reaction (PCR) for retroviral DNA.

Statistical analysis

The probability of significant difference between groups of cultures wasdetermined by comparing the cumulative cell production values from theexperimental samples to the matched control cultures using a pairedt-test. Statistical significant was taken at the 5% level.

Results:

Retroviral Infection Using SAX Retrovirus

Kinetics of cell production in LTBMCs infected with SAX retrovirus

Cell production in retrovirally-infected cultures is an indicator of thelikelihood of retroviral infection and is therefore a useful parameterto measure. Retroviral integration into the target cell genome is onlythought to occur during cell division. For this reason, increasedculture productivity increases the probability of stem cell mitosis andthereby increases the probability of retroviral infection. The highestcell production occurred in the cultures with supernatant virus additionsupplemented with IL-3+GM-CSF and IL-3+GM-CSF+IL-1α which producedincreasing number of cells through 4 weeks in culture. The LTBMCsco-cultured with the SAX virus packaging cells produced more cells thanthe supernatant addition cultures at week two, although cell productiondecreased after week 2.

Analysis of retroviral infection in LTBMCs with supernatant SAX virusaddition

The percentage of progenitor cells surviving in high G418 concentrationvaried from 2 to 50% in the cultures supplemented with IL-3+GM-CSF orIL-3+GM-CSF+IL-1α during the first four to six weeks in culture. By 10weeks in culture (8 weeks after virus addition had ended) 43% of thenumber of progenitor cells that were clonable to hematopoietic coloniessurvived exposure to G418. This indicates that these progenitor cellshad been rendered G418 resistant by virtue of containing the G418resistance gene transferred by the retrovirus to stem cells present inthe culture during the initial 14 day infection period. The rapidlyperfused cultures not supplemented with HGFs had an average 12%progenitor cell survival in high G418 concentration between weeks 8 and11. At culture termination at 11 weeks, the stromal layer of theIL-3+GM-CSF supplemented cultures was trypsinized and 17% of progenitorcells that were adherent to the stroma survived in high G418. Thissuggests that a significant percentage of adherent progenitor cells werealso infected with the SAX virus.

Analysis of retroviral infection in LTBMCs co-cultured with irradiatedSAX virus packaging cells

The percentage of progenitor cells surviving in G418 when co-culturedwith irradiated SAX cells varied between 0 and 36%. The cultures notsupplemented with HGFs produced CFU-GM that survived in high G418concentration only between weeks 4-7. After week 7, no CFU-GM wereproduced In these cultures that survived in high G418!, suggesting thatlittle or no infection of stem cells occurred. The LTBMCs supplementedwith IL-3+GM-CSF and co-cultured with irradiated SAX cells for 2.5 weeksproduced high percentages of CFU-GM that survived in 0.8 mg/ml G418 atweeks 4, 5 and 8. However, at week 10, these cultures failed to produceCFU-GM that were resistant to G418. This suggests that little or noinfection of stem cells occurred in these cultures or that the stemcells may have differentiated or died.

Retroviral infection using DC-29 retrovirus

Kinetics of cell production in LTBMCs infected with DC-29 retrovirus

The number of cells produced in the cultures infected with DC-29retroviral supernatant was HGF dependent. The cultures supplemented withIL-3+GM-CSF+Epo produced between 1.5-4×10⁶ cells on a weekly basisthroughout the 10 weeks of culture. The cultures supplemented withIL-3+GM-CSF+Epo+MGF were more prolific, while the cultures supplementedwith Hybrikine+Epo resulted in the highest cell production.Interestingly, the control cultures supplemented with IL-3+GM-CSF+Epobut not receiving DC-29 retroviral supernatant additions were lessprolific than the similar cultures receiving the DC-29 retroviralsupernatant. Cell production in the IL-3+GM-CSF+Epo, IL-3+GM-CSF+Epo+MGFand Hybrikine+Epo cultures (with virus addition) was significantlyhigher than the control culture (IL-3+GM-CSF+Epo, no virus addition) atthe 5%, 1% and 1% level of significance, respectively. A part of theincreased production in the cultures with retroviral supernatantaddition may be due to the presence of a growth factor(s) such as MGF(c-kit ligand) which is known to be produced by the 3T3-based packagingcell line.

Analysis of retroviral infection in LTBMCs with supernatant DC-29 virusaddition

The efficiency of retroviral infection was assessed by CFU-GM survivalin 1.6 mg/ml G418, a concentration that killed all bone marrow cellsprior to retroviral infection. The average percent of CFU-GM survivingat week 8 (6 weeks after infection) was high in all infected cultures,Table 5.

                                      TABLE 5                                     __________________________________________________________________________    Percent of CFU-GM surviving in 1.6 mg/ml G418 after infection with DC-29      retrovirus as                                                                 a function of HGF supplementation. The cultures were infected with DC-29      retroviral                                                                    containing supernatent for the initial 2 weeks in culture.                    IL-3 + GM-CSF + Epo                                                                              IL-3 + GM-CSF + Epo + MGF                                                                    Hibrikine + Epo                             with virus         with virus     with virus                                  week                                                                              1  2  3  ±dev.                                                                         ave.                                                                             1  2  3  ±dev.                                                                         ave.                                                                             1  2  3  ±dev.                                                                         ave.                            __________________________________________________________________________    2   4.70                                                                             2.40                                                                             5.60                                                                             1.6                                                                              4.2                                                                              3.70                                                                             21.6                                                                             19.5                                                                             9.7                                                                              1.49                                                                             9.00                                                                             6.20                                                                             18.4                                                                             6.4                                                                              11.2                            4   2.5                                                                              0.0                                                                              4.6                                                                              2.5                                                                              2.4                                                                              8.1                                                                              6.0                                                                              16.1                                                                             3.3                                                                              10.1                                                                             9.0                                                                              6.2                                                                              18.4                                                                             5.4                                                                              4.4                             6   0.0                                                                              0.0                                                                              0.0                                                                              0.0                                                                              0.0                                                                              1.4                                                                              1.7                                                                              4.0                                                                              1.4                                                                              2.4                                                                              0.0                                                                              0.0                                                                              0.0                                                                              0.0                                                                              0.0                             8   0.0                                                                              22.2                                                                             *  12.8                                                                             7.4                                                                              0.0                                                                              14.5                                                                             11.5                                                                             3.6                                                                              9.0                                                                              21.6                                                                             14.7                                                                             10.7                                                                             5.5                                                                              15.7                            10  *  0.0                                                                              *  0.0                                                                              0.0                                                                              8.2                                                                              0.0                                                                              0.0                                                                              4.7                                                                              2.7                                                                              0.0                                                                              0.0                                                                              0.0                                                                              0.0                                                                              0.0                             10†                                                                        2.6                                                                              0.0                                                                              *  1.5                                                                              0.9                                                                              7.7                                                                              0.0                                                                              *  4.4                                                                              2.6                                                                              0.0                                                                              0.0                                                                              0.0                                                                              0.0                                                                              0.0                             __________________________________________________________________________     *N/A because no CFUGM were counted in 0.0 mg/ml G418.                         † % CFUGM surviving in 1.6 mg/ml G418 from the trypsinized stroma      in each culture at experiment termination                                     % survival was calculated as the (number of CFUGM at 1.6 mg/ml G418 - ave     number of CFUGM at 1.6 mg/ml G418 in uninfected cultures) divided by          (number of CFUGM at 0.0 mg/ml G418) * 100                                

Addition of MGF to the combination of IL-3+GM-CSF+Epo appears to haveincreased infection efficiency at weeks 8-10 in culture, with one of thecultures containing 7.7% G418 resistant colonies. The data at week 8suggests that the cultures supplemented with Hybrikine+Epo had a highinfection efficiency, although the data at week 10 suggests noinfection. At week 10, note that the number of CFU-GM removed fromseveral cultures decreased to where few or no CFU-GM would be expectedbe seen at approximately 10% infection. In addition, 1.5 mg/ml G418 isan extremely high antibiotic dose, enough to likely overwhelm theefficacy of the G418 resistance gene transfected at even slightlysuboptimal levels. Thus, the efficiency of gene transfer into thehematopoietic stem cells in these cultures was at least 7.7% in somesamples, and perhaps as high as 45% or higher.

Kinetics of nonadherent progenitor cell production

Because analysis of retroviral infection in these experiments depends onassaying for progenitor cells to infer stem cell existence, infection,and cycling, it is important to determine the effect of HGFs onprogenitor cell production in culture. Also, in this retroviralexperiment, MGF and Hybrikine are being used in combination with otherHGFs. Both MGF and Hybrikine have not been used in rapidly perfused HGFsupplemented LTBMCs, and it is therefore necessary to determine theireffects on hematopoiesis.

Progenitor cell production was strongly dependent on HGFsupplementation. The number of CFU-GM removed from the cultures is shownin Table 6.

                  TABLE 6                                                         ______________________________________                                        The cumulative number of progenitor cells                                     removed in DC-29, HGF supplemented rapidly                                    perfused human long-term bone marrow cultures.                                                             Total Number                                                                  of CFU--GM                                       Culture            Virus Added                                                                             Removed                                          ______________________________________                                        Innoculum          N/A        900                                             Control            No        1300                                             (IL - 3 + GM - CSF + Epo)                                                     IL - 3 + GM - CSF + Epo                                                                          Yes       2800                                             IL - 3 + GM - CSF + Epo + MGF                                                                    Yes       5400                                             Hybrikine + Epo    Yes       5000                                             ______________________________________                                         Every second weekly sampling from the cultures was assayed for CFU--GM an     the non assayed values were estimated by linear interpolation between two     known data points. The known and interpolated values were summed to           approximate the total number of CFU--GM removed from culture.            

Addition of retroviral supernatant increased progenitor cell removal2.2-fold over no addition of retroviral supernatant. The increase in thenumber of CFU-GM removed in the cultures supplemented with retroviralsupernatant and IL-3+GM-CSF+Epo+MGF or Hybrikine+Epo was 4.2 and3.8-fold greater then the uninfected control, respectively. Removal ofCFU-GM was statistically greater, at the 1% level of significance, inall viral supplemented cultures when compared to the number of CFU-GMinoculated.

Progenitor cells production in culture

A population balance on the CFU-GM compartment shows that the additionof MGF or Hybrikine to cultures supplemented with IL-3+GM-CSF+Epo have asignificant positive effect on the CFU-GM pool. The addition of MGF tothe combination of IL-3+GM-CSF+Epo increased CFU-GM removal 1.9-fold andCFU-GM differentiation 0.5-fold compared to similar cultures notsupplemented with MGF, Table 7.

                  TABLE 7                                                         ______________________________________                                        The cumulative production of CFU--GM removed and                              differentiated in growth factor supplemented DC-29                            retroviral infected rapidly perfused human long-term                          bone marrow cultures.                                                                              Number of                                                                              Estimated                                                  Number    GM       number                                                     of CFU--GM                                                                              cells    of CFU--GM                                                                            Total                                              removed   removed  that differ-                                                                          number of                               Culture    (plated)  (× 10.sup.-6)                                                                    entiated                                                                              CFU--GM                                 ______________________________________                                        Innoculum  (900)     (0)      (0)     (900)                                   control    1300      1.1      1100    2400                                    (IL - 3 + GM -                                                                CSF + Epo,                                                                    no virus added)                                                               (IL - 3 + GM -                                                                           2800      1.8      1800    4600                                    CSF + Epo                                                                     (virus added)                                                                 (IL - 3 + GM -                                                                           5400      3.1      3100    8400                                    CSF + Epo + MGF                                                               (virus added)                                                                 Hybrikine + Epo                                                                          5000      6.1      6100    11,100                                  (virus added)                                                                 ______________________________________                                         Every second weekly sampling from the cultures was assayed for CFU--GM an     the non assayed values were estimated by linear interpolation between two     known data points. The known and interpolated values were summed to           approximate the total number of CFU--GM removed from culture.            

The combination of Hybrikine+Epo had a very pronounced effect on CFU-GMproduction and differentiation. Hybrikine+Epo induced a 1.8-foldincrease in CFU-GM removal and over a 3-fold increase in CFU-GMdifferentiation compared to the previous optimum cultures supplementedwith IL-3+GM-CSF+Epo. Also, Hybrikine+Epo induced production of almosttwice the number of granulocytes and macrophages than did thecombination of IL-3+GM-CSF+Epo+MGF. This indicates that Hybrikine is apotent inducer of the granulocyte macrophage lineages.

Analysis of neutrophils produced from stem cells infected with CD18encoding retrovirus

CD18 deficient bone marrow was enriched for early hematopoietic cells asdescribed above, and then cultured for 14 days with 50% daily mediumexchange supplemented with 1.0 ng/ml/day GM-CSF and 1.0 ng/ml/day IL-3and 40 U/ml/day IL-1α, and with CD18 retroviral producer linesupernatant. From day 15 on, the cells were cultured under the sameconditions without addition of retroviral supernatant. Nonadherent cellswere removed from the cultures weekly and analyzed for the presence ofcell surface CD18 by flow cytometry using a biotinylated anti-CD18monoclonal antibody by standard methods (Updyke et al Meth. Enzymol.(1986) 121:717-725). Whereas CD18 deficient bone marrow cells failed toexpress any cell surface CD18 protein by this assay, neutrophils andmonocytes that arose from the retrovirally infected cultures did expresscell surface CD18. In triplicate cultures, expression of cell surfaceCD18 was 3.5%/5%/2% at 6 weeks and 11%/28%/3% at 11 weeks. Since theneutrophils and monocytes present in the cultures at 11 weeks arose formCFU-GM progenitor cells only 10-14 days earlier, these data indicatethat human hematopoietic stem cells were successfully and stablytransfected with the recombinant retrovirus during the first two weeksof the culture.

In interpreting the present results it is important to recognize thatalthough several groups have demonstrated retroviral-mediated genetransfer into human hematopoietic progenitor cells, gene transfer intohuman hematopoietic stem cells has not been shown. The rapid decay ofcell production in traditional, slowly perfused human LTBMCs limitedinfection determination due to the absence of progenitor cells requiredfor the assay.

Retroviral infection in the present studies was initially assessed bygrowing cells removed from the LTBMCs in methylcellulose in the presenceof the mammalian cell antibiotic G418. Progenitor cells infected withthe SAX or DC-29 retroviruses and expressing the NEO product will beable to survive and form colonies in high concentrations of G418,whereas noninfected cells will die in high concentrations of G418. Also,production of progenitor cells that survive in high concentrations ofG418, six or more weeks after virus addition has ended, requires thosecells to have recently differentiated from a more primitive cell (a stemcell) and therefore suggests that stem cells were infected. Similarly,the CD18 expressed on the surface of neutrophils and monocytes producedduring the 11th week of culture requires that a primitive hematopoieticstem cell was infected during the first 2 weeks of the cultures, becauseall mature cells, precursors, and clonogenic progenitors present duringthe infection period had died by 4-5 weeks in the culture.

The analysis for retroviral infection used here can underestimate thepercentage of cells infected with the retrovirus due to insufficientexpression of the NEO gene product. Under-expression of the transferredgene product has been shown to be a problem in human and primate models.Therefore, the percentage of progenitor cells infected in this study isprobably a conservative estimate.

The percentage of progenitor cells that survived in high G418concentration was approximately 40% at week 10 in the cultures infectedSAX retrovirus supernatant and supplemented with IL-3+GM-CSF±IL-1α.These initial results show that a high percentage of stem cells wereinfected in the cultures supplemented with retroviral supernatant duringthe first two weeks in culture.

The percentage of progenitor cells infected with the DC-29 retroviruswas high (0-21%) in the IL-3+GM-CSF+Epo+MGF and Hybrikine+Epo culturesduring the initial 4 weeks after viral addition had ended. This highlevel of progenitor cell infection was probably due to direct infectionof progenitor and primitive cells by the retrovirus. The percentage ofprogenitor cells surviving in high G418 concentration decreased 4 weeksafter viral addition had ended, but rebounded 2 weeks later to 0-22%survival in high G418 concentration.

The production of G418 resistant progenitor cells in the DC-29experiment may underestimate the percentage of progenitor cell actuallyinfected with the DC-29 retrovirus. The high concentration of G418 usedto select for infected colonies (1.6 mg/ml of G418) is twice as high asthat used for the SAX infection experiment and requires high expressionlevels of the NEO gene product, neomycin phosphotransferase, to survive.

Interestingly, in the LTBMCs supplemented with either the SAX or DC-29retrovirus containing supernatant, the HGFs IL-3+GM-CSF±Epo, IL-1α, orMGF or the combination of Hybrikine+Epo, the percent of progenitor cellssurviving in G418 increased 6-8 weeks after viral addition ended.Although the mechanism for the increase in the percentage of G418surviving progenitor cells towards the latter stages of the culture arenot known one possibility is that stem cells that were infected duringthe first two weeks in culture became more active as the cultureprogressed. This would effectively increase the percentage of cellssurviving in G418. Another possibility is that expression of the NEOgene may have increased in progenitor cells produced late in culture dueto differentiation from a stem cell which had a different and betterexpressible integration site than did progenitor cells transfecteddirectly during the initial infection culture period. Therefore, thehigh level of progenitor cell survival late in culture although possiblydue to several causes, does strongly suggest that stem cells wereinfected in these LTBMCs.

In sum, these data document that under the culture conditions disclosedin the present invention, hematopoietic stem cells were proliferating inthe cultures, permitting the incorporation of retrovirally transferredgenetic material into these cells. Progenitors were continuously andactively produced from these stem cells, and these many of theseprogenitors contained and expressed the transfected genes. These dataindicate that genetically modified human hematopoietic stem cells werepresent and proliferating in these cultures.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed as new and desired to be secure by Letters Patent of theUnited States is:
 1. An ex vivo human stem cell composition in which asubstantial number of human stem cells originating from a humanhematopoietic system have undergone ex vivo cell division as a result ofthe conditions in a medium-exchange ex vivo culture, comprising stablegenetically transformed human stem cells originating from a humanhematopoietic system.
 2. An ex vivo human hematopoietic or stromal stemcell composition in which a substantial number of human hematopoietic orstromal stem cells have undergone ex vivo cell division as a result ofthe conditions in a medium-exchange ex vivo culture, comprising stablegenetically transformed human hematopoietic or stromal stem cells.
 3. Anex vivo human bone marrow stem cell composition in which a substantialnumber of human bone marrow stem cells have undergone ex vivo celldivision as a result of the conditions in a medium-exchange ex vivoculture, comprising stable genetically transformed human bone marrowstem cells.
 4. The ex vivo human stem cell composition of claim 1,further comprising stable, genetically transformed human progenitorcells.
 5. The ex vivo human stem cell composition of claim 1, comprisingat least one exogenous growth factor or cytokine.
 6. The ex vivo humanstem cell composition of claim 1, obtained by culturing human stem cellsobtained from a human hematopoietic system in the presence of a genetransfer vector and in a liquid culture medium which is replaced at arate which is substantially continuous and provides ex vivo human stemcell division, while maintaining said culture under physiologicallyacceptable conditions.
 7. The ex vivo human stem cell composition ofclaim 1, obtained by culturing human stem cells obtained from a humanhematopoietic system in the presence of a gene transfer vector and in aliquid culture medium which is replaced at a rate of at least 50% dailyreplacement, while maintaining said culture under physiologicallyacceptable conditions.
 8. The ex vivo human stem cell composition ofclaim 1, obtained by culturing human stem cells obtained from a humanhematopoietic system in the presence of a gene transfer vector and in aliquid culture medium which is replaced at a rate related to the celldensity of said culture, said rate being equal to a rate of 50 to 100%daily replacement for a cell density of from 1×10⁴ to 1×10⁷ cells per mlof culture.
 9. The ex vivo human stem cell composition of claim 1,wherein said composition comprises at least one member selected from thegroup consisting of human peripheral blood mononuclear cells, human bonemarrow cells, human fetal liver cells, and human cord blood cells. 10.The ex vivo human stem cell composition of claim 9, wherein said humanperipheral blood mononuclear cells, human bone marrow cells, human fetalliver cells, and human cord blood cells, are enriched for said humanstem cells obtained from a human hematopoietic system.
 11. The ex vivohuman stem cell composition of claim 1, wherein said compositioncomprises at least one member selected from the group consisting ofhuman peripheral blood mononuclear cells, human bone marrow cells, humanfetal liver cells, and human cord blood cells; said composition alsocomprising human stromal cells.
 12. The ex vivo human stem cellcomposition of claim 1, wherein said human stem cells are found in ahuman peripheral blood.
 13. The ex vivo human stem cell composition ofclaim 1, wherein said human stem cells are found in a human fetal liver.14. The ex vivo human stem cell composition of claim 1, wherein saidhuman stem cells are found in a human umbilical cord blood.
 15. The exvivo human stem cell composition of claim 1, comprising stablegenetically transformed human stromal lineage cells.
 16. The ex vivohuman stem cell composition of claim 1, under static medium conditions.17. The ex vivo human stem cell composition of claim 1, undermedium-exchange conditions.
 18. The ex vivo human stem cell compositionof claim 2, further comprising stable, genetically transformed humanhematopoietic or stromal progenitor cells.
 19. The ex vivo human stemcell composition of claim 2, comprising at least one exogenous growthfactor or cytokine.
 20. The ex vivo human stem cell composition of claim2, obtained by culturing human hematopoietic or stromal stem cells inthe presence of a gene transfer vector and in a liquid culture mediumwhich is replaced at a rate which is substantially continuous andprovides ex vivo human hematopoietic or stromal stem cell division,while maintaining said culture under physiologically acceptableconditions.
 21. The ex vivo human hematopoietic or stromal stem cellcomposition of claim 2, obtained by culturing human hematopoietic orstromal stem cells in the presence of a gene transfer vector and in aliquid culture medium which is replaced at a rate of at least 50% dailyreplacement, while maintaining said culture under physiologicallyacceptable conditions.
 22. The ex vivo human hematopoietic or stromalstem cell composition of claim 2, obtained by culturing humanhematopoietic or stromal stem cells in the presence of a gene transfervector and in a liquid culture medium which is replaced at a raterelated to the cell density of said culture, said rate being equal to arate of 50 to 100% daily replacement for a cell density of from 1×10⁴ to1×10⁷ cells per ml of culture.
 23. The ex vivo human hematopoietic orstromal stem cell composition of claim 2, wherein said compositioncomprises at least one member selected from the group consisting ofhuman peripheral blood mononuclear cells, human bone marrow cells, humanfetal liver cells, and human cord blood cells.
 24. The ex vivo humanhematopoietic or stromal stem cell composition of claim 23, wherein saidhuman peripheral blood mononuclear cells, human bone marrow cells, humanfetal liver cells, and human cord blood cells, are enriched for saidhuman hematopoietic or stromal stem cells.
 25. The ex vivo humanhematopoietic or stromal stem cell composition of claim 2 wherein saidcomposition comprises at least one member selected from the groupconsisting of human peripheral blood mononuclear cells, human bonemarrow cells, human fetal liver cells, and human cord blood cells; saidcomposition also comprising human stromal cells.
 26. The ex vivo humanhematopoietic or stromal stem cell composition of claim 2, wherein saidhuman hematopoietic or stromal stem cells are found in a humanperipheral blood.
 27. The ex vivo human hematopoietic or stromal stemcell composition of claim 2, wherein said human hematopoietic or stromalstem cells are found in a human fetal liver.
 28. The ex vivo humanhematopoietic or stromal stem cell composition of claim 2, wherein saidhuman hematopoietic or stromal stem cells are found in a human umbilicalcord blood.
 29. The ex vivo human hematopoietic or stromal stem cellcomposition of claim 2, comprising stable genetically transformed humanstromal lineage cells.
 30. The ex vivo human hematopoietic or stromalstem cell composition of claim 2, comprising stable genetically,transformed human hematopoietic lineage cells.
 31. The ex vivo humanhematopoietic or stromal stem cell composition of claim 2, under staticmedium conditions.
 32. The ex vivo human hematopoietic or stromal stemcell composition of claim 2, under medium-exchange conditions.
 33. Theex vivo human bone marrow stem cell composition of claim 3, furthercomprising stable, genetically transformed human bone marrow progenitorcells.
 34. The ex vivo human bone marrow stem cell composition of claim3, comprising at least one exogenous growth factor or cytokine.
 35. Theex vivo human bone marrow stem cell composition of claim 3, obtained byculturing human bone marrow stem cells in the presence of a genetransfer vector and in a liquid culture medium which is replaced at arate which is substantially continuous and provides ex vivo human bonemarrow stem cell division, while maintaining said culture underphysiologically acceptable conditions.
 36. The ex vivo human bone marrowstem cell composition of claim 3, obtained by culturing human bonemarrow stem cells in the presence of a gene transfer vector and in aliquid culture medium which is replaced at a rate of at least 50% dailyreplacement, while maintaining said culture under physiologicallyacceptable conditions.
 37. The ex vivo human bone marrow stem cellcomposition of claim 3, obtained by culturing human bone marrow stemcells in the presence of a gene transfer vector and in a liquid culturemedium which is replaced at a rate related to the cell density of saidculture, said rate being equal to a rate of 50 to 100% daily replacementfor a cell density of from 1×10⁴ to 1×10⁷ cells per ml of culture. 38.The ex vivo human bone marrow stem cell composition of claim 3, whereinsaid composition comprises at least one member selected from the groupconsisting of human peripheral blood mononuclear cells, human bonemarrow cells, human fetal liver cells, and human cord blood cells. 39.The ex vivo human bone marrow stem cell composition of claim 38, whereinsaid human peripheral blood mononuclear cells, human bone marrow cells,human fetal liver cells, and human cord blood cells, are enriched forsaid human bone marrow stem cells.
 40. The ex vivo human bone marrowstem cell composition of claim 3, wherein said composition comprises atleast one member selected from the group consisting of human peripheralblood mononuclear cells, human bone marrow cells, human fetal livercells, and human cord blood cells; said composition also comprisinghuman stromal cells.
 41. The ex vivo human bone marrow stem cellcomposition of claim 3, wherein said human bone marrow stem cells arefound in a human peripheral blood.
 42. The ex vivo human bone marrowstem cell composition of claim 3, wherein said human bone marrow stemcells are found in a human fetal liver.
 43. The ex vivo human bonemarrow stem cell composition of claim 3, wherein said human bone marrowstem cells are found in a human umbilical cord blood.
 44. The ex vivohuman bone marrow stem cell composition of claim 3, comprising stablegenetically transformed human hematopoietic or stromal lineage cells.45. The ex vivo human bone marrow stem cell composition of claim 3,under static medium conditions.
 46. The ex vivo human bone marrow stemcell composition of claim 3, under medium-exchange conditions.
 47. Amethod for obtaining ex vivo human stem cell stable genetictransformation comprising exposing, to a gene transfer vector, humanstem cells originating from a human hematopoietic system and cultured ina liquid culture medium which is replaced at a rate which issubstantially continuous and provides ex vivo human stem cell division,while maintaining said culture under physiologically acceptableconditions.
 48. A method for obtaining ex vivo human stem cell stablegenetic transformation comprising exposing, to a gene transfer vector,human stem cells originating from a human hematopoietic system andcultured in a liquid culture medium which is replaced at a rate equal toa rate of 50 to 100% daily replacement for a cell density of from 1×10⁴to 1×10⁷ cells per ml of culture, while maintaining said culture underphysiologically acceptable conditions, and obtaining stable geneticallytransformed human stem cells originating from a human hematopoieticsystem.
 49. A method for obtaining ex vivo human stem cell stablegenetic transformation comprising exposing, to a gene transfer vector,human stem cells originating from a human hematopoietic system andcultured in a liquid culture medium which is replaced at a rate of atleast 50% daily replacement while maintaining said culture underphysiologically acceptable conditions, and obtaining stable geneticallytransformed human stem cells originating from a human hematopoieticsystem.
 50. A method for obtaining ex vivo human hematopoietic orstromal stem cell stable genetic transformation comprising exposing, toa gene transfer vector, human hematopoietic or stromal stem cellscultured in a liquid culture medium which is replaced at a rate which issubstantially continuous and provides ex vivo human stem cell division,while maintaining said culture under physiologically acceptableconditions.
 51. A method for obtaining ex vivo human hematopoietic orstromal stem cell stable genetic transformation comprising exposing, toa gene transfer vector, human hematopoietic or stromal stem cellscultured in a liquid culture medium which is replaced at a rate equal toa rate of 50 to 100% daily replacement for a cell density of from 1×10⁴to 1×10⁷ cells per ml of culture, while maintaining said culture underphysiologically acceptable conditions, and obtaining stable geneticallytransformed human hematopoietic or stromal stem cells.
 52. A method forobtaining ex vivo human hematopoietic or stromal stem cell stablegenetic transformation comprising exposing, to a gene transfer vector,human hematopoietic or stromal stem cells cultured in a liquid culturemedium which is replaced at a rate of at least 50% daily replacementwhile maintaining said culture under physiologically acceptableconditions, and obtaining stable genetically transformed humanhematopoietic or stromal stem cells.
 53. A method for obtaining ex vivohuman bone marrow stem cell stable genetic transformation comprisingexposing, to a gene transfer vector, human bone marrow stem cellscultured in a liquid culture medium which is replaced at a rate which issubstantially continuous and provides ex vivo human bone marrow stemcell division, while maintaining said culture under physiologicallyacceptable conditions.
 54. A method for obtaining ex vivo human bonemarrow stem cell stable genetic transformation comprising exposing, to agene transfer vector, human bone marrow stem cells cultured in a liquidculture medium which is replaced at a rate related to the cell densityof said culture, said rate being equal to a rate of 50 to 100% dailyreplacement for a cell density of from 1×10⁴ to 1×10⁷ cells per ml ofculture, while maintaining said culture under physiologically acceptableconditions, and obtaining stable genetically transformed human bonemarrow stem cells.
 55. A method for obtaining ex vivo human bone marrowstem cell stable genetic transformation comprising exposing, to a genetransfer vector, human bone marrow stem cells cultured in a liquidculture medium which is replaced at a rate of at least 50% dailyreplacement while maintaining said culture under physiologicallyacceptable conditions, and obtaining stable genetically transformedhuman bone marrow stem cells.
 56. The method of claim 47, comprisingusing a liquid culture medium having at least one exogenous growthfactor or cytokine.
 57. The method of claim 50, comprising using aliquid culture medium having at least one exogenous growth factor orcytokine.
 58. The method of claim 53, comprising using a liquid culturemedium having at least one exogenous growth factor or cytokine.
 59. Themethod of claim 47, wherein said medium is replaced continuously. 60.The method of claim 59, wherein the replacement of said medium comprisesperfusing fresh medium through at least part of the mass of said humanstem cells obtained from a human hematopoietic system.
 61. The method ofclaim 47, wherein said medium is replaced periodically.
 62. The methodof claim 61, wherein the replacement of said medium comprises perfusingfresh medium through at least part of the mass of said human stem cellsobtained from a human hematopoietic system.
 63. The method of claim 47,wherein IL-3 and GM-CSF are added to said medium, each at a rate of 0.1to 100 ng ml⁻¹ day⁻¹.
 64. The method of claim 47, wherein IL-3 andGM-CSF are added to said medium, each at a rate of 0.5 to 10 ng ml⁻¹day⁻¹.
 65. The method of claim 47, wherein IL-3 and GM-CSF are added tosaid medium, each at a rate of 1 to 2 ng ml⁻¹ day⁻¹.
 66. The method ofclaim 47, wherein steel factor is added to said medium at a rate of from1 to 100 ng ml⁻¹ day⁻¹, or IL-1α is added to said medium at a rate offrom 10 to 100 U ml⁻¹ per 3 to 5 day period.
 67. The method of claim 66,wherein steel factor is added to said medium.
 68. The method of claim66, wherein IL-1α is added to said medium at a rate of from 10 to 100 Uml⁻¹ per 3 to 5 day period.
 69. The method of claim 66, wherein bothsteel factor and IL-1α are added to said medium.
 70. The method of claim47, wherein said medium comprises animal sera or plasma.
 71. The methodof claim 47, wherein said medium comprises human sera or plasma.
 72. Themethod of claim 47, comprising maintaining glucose concentration in saidmedium in the range of from 5 to 20 mM, lactate concentration in saidmedium below about 35 mM, glutamine concentration in said medium in therange of from 1 to 3 mM, and ammonia concentration in said medium below2.5 mM.
 73. The method of claim 47, wherein said gene transfer vector isa retrovirus.
 74. The method of claim 47, comprising culturing saidhuman stem cells originating from a human hematopoietic system in thepresence of a retroviral packaging cell line supernatant.
 75. The methodof claim 47, comprising culturing said human stem cells originating froma human hematopoietic system in the presence of a retroviral packagingcell line.
 76. The method of claim 47, wherein the population of humanprogenitor cells originating from a human hematopoietic system, in saidculture, expands.
 77. The method of claim 47, wherein said culturemedium contains at least one member selected from the group consistingof human peripheral blood mononuclear cells, human bone marrow cells,human fetal liver cells, and human cord blood cells.
 78. The method ofclaim 77, wherein said human peripheral blood mononuclear cells, humanbone marrow cells, human fetal liver cells, and human cord blood cells,are enriched for said human stem cells originating from a humanhematopoietic system.
 79. The method of claim 47, wherein said culturedhuman stem cells originating in a human hematopoietic system producestable, genetically transformed human progenitor cells.
 80. The methodof claim 47, wherein stable genetically transformed human leukocytes,platelets, lymphocytes, osteoblasts or osteoclasts are obtained.
 81. Themethod of claim 47, wherein said cells are cultured in the presence ofan immobilized antibody specific to said human stem cells originatingfrom a human hematopoietic system.
 82. The method of claim 47, whereinsaid human stem cells originating from a human hematopoietic system arefound in a human peripheral blood.
 83. The method of claim 47, whereinsaid human stem cells originating from a human hematopoietic system arefound in a human fetal liver.
 84. The method of claim 47, wherein saidhuman stem cells originating from a human hematopoietic system are foundin a human umbilical cord blood.
 85. The method of claim 50, whereinsaid medium is replaced continuously.
 86. The method of claim 85,wherein the replacement of said medium comprises perfusing fresh mediumthrough at least part of the mass of said human hematopoietic or stromalstem cells.
 87. The method of claim 50, wherein said medium is replacedperiodically.
 88. The method of claim 87, wherein the replacement ofsaid medium comprises perfusing fresh medium through at least part ofthe mass of said human hematopoietic or stromal stem cells.
 89. Themethod of claim 50, wherein IL-3 and GM-CSF are added to said medium,each at a rate of 0.1 to 100 ng ml⁻¹ day⁻¹.
 90. The method of claim 50,wherein IL-3 and GM-CSF are added to said medium, each at a rate of 0.5to 10 ng ml⁻¹ day⁻¹.
 91. The method of claim 50, wherein IL-3 and GM-CSFare added to said medium, each at a rate of 1 to 2 ng ml⁻¹ day⁻¹. 92.The method of claim 51, wherein steel factor is added to said medium ata rate of from 1 to 100 ng ml⁻¹ day⁻¹, or IL-1α is added to said mediumat a rate of from 10 to 100 U ml⁻¹ per 3 to 5 day period.
 93. The methodof claim 92, wherein steel factor is added to said medium.
 94. Themethod of claim 92, wherein IL-1α is added to said medium at a rate offrom 10 to 100 U ml⁻¹ per 3 to 5 day period.
 95. The method of claim 92,wherein both steel factor and IL-1α are added to said medium.
 96. Themethod of claim 50, wherein said medium comprises animal sera or plasma.97. The method of claim 50, wherein said medium comprises human sera orplasma.
 98. The method of claim 50, comprising maintaining glucoseconcentration in said medium in the range of from 5 to 20 mM, lactateconcentration in said medium below about 35 mM, glutamine concentrationin said medium in the range of from 1 to 3 mM, and ammonia concentrationin said medium below 2.5 mM.
 99. The method of claim 50, wherein saidgene transfer vector is a retrovirus.
 100. The method of claim 50,comprising culturing said human hematopoietic or stromal item cells inthe presence of a retroviral packaging cell line supernatant.
 101. Themethod of claim 50, comprising culturing said human hematopoietic orstromal stem cells in the presence of a retroviral packaging cell line.102. The method of claim 50, wherein the population of humanhematopoietic or stromal progenitor cells, in said culture, expands.103. The method of claim 50, wherein said culture medium contains atleast one member selected from the group consisting of human peripheralblood mononuclear cells, human bone marrow cells, human fetal livercells, and human cord blood cells.
 104. The method of claim 103, whereinsaid human peripheral blood mononuclear cells, human bone marrow cells,human fetal liver cells, and human cord blood cells, are enriched forsaid human hematopoietic or stromal stem cells.
 105. The method of claim50, wherein said cultured human hematopoietic or stromal stem cellsproduce stable, genetically transformed human hematopoietic or stromalprogenitor cells.
 106. The method of claim 50, wherein said culturedhuman hematopoietic or stromal stem cells produce stable, geneticallytransformed human hematopoietic lineage cells.
 107. The method of claim50, wherein said cultured human hematopoietic or stromal stem cellsproduce stable, genetically transformed human stromal lineage cells.108. The method of claim 50, wherein stable genetically transformedhuman leukocytes, platelets, lymphocytes, osteoblasts or osteoclasts areobtained.
 109. The method of claim 50, wherein said cells are culturedin the presence of an immobilized antibody specific to said humanhematopoietic or stromal stem cells.
 110. The method of claim 50,wherein said human hematopoietic or stromal stem cells are found in ahuman peripheral blood.
 111. The method of claim 50, wherein said humanhematopoietic or stromal stem cells are found in a human fetal liver.112. The method of claim 50, wherein said human stem cells are found ina human umbilical cord blood.
 113. The method of claim 53, wherein saidmedium is replaced continuously.
 114. The method of claim 113, whereinthe replacement of said medium comprises perfusing fresh medium throughat least part of the mass of said human bone marrow stem cells.
 115. Themethod of claim 53, wherein said medium is replaced periodically. 116.The method of claim 115, wherein the replacement of said mediumcomprises perfusing fresh medium through at least part of the mass ofsaid human bone marrow stem cells.
 117. The method of claim 53, whereinIL-3 and GM-CSF are added to said medium, each at a rate of 0.1 to 100ng ml⁻¹ day⁻¹.
 118. The method of claim 53, wherein IL-3 and GM-CSF areadded to said medium, each at a rate of 0.5 to 10 ng ml⁻¹ day⁻¹. 119.The method of claim 53, wherein IL-3 and GM-CSF are added to saidmedium, each at a rate of 1 to 2 ng ml⁻¹ day⁻¹.
 120. The method of claim53, wherein steel factor is added to said medium at a rate of from 1 to100 ng ml⁻¹ day⁻¹, or IL-1α is added to said medium at a rate of from 10to 100 U ml⁻¹ per 3 to 5 day period.
 121. The method of claim 120,wherein steel factor is added to said medium.
 122. The method of claim120, wherein IL-1α is added to said medium at a rate of from 10 to 100 Uml⁻¹ per 3 to 5 day period.
 123. The method of claim 120, wherein bothsteel factor and IL-1α are added to said medium.
 124. The method ofclaim 53, wherein said medium comprises animal sera or plasma.
 125. Themethod of claim 53, wherein said medium comprises human sera or plasma.126. The method of claim 53, comprising maintaining glucoseconcentration in said medium in the range of from 5 to 20 mM, lactateconcentration in said medium below about 35 mM, glutamine concentrationin said medium in the range of from 1 to 3 mM, and ammonia concentrationin said medium below 2.5 mM.
 127. The method of claim 53, wherein saidgene transfer vector is a retrovirus.
 128. The method of claim 53,comprising culturing said human bone marrow stem cells in the presenceof a retroviral packaging cell line supernatant.
 129. The method ofclaim 53, comprising culturing said human bone marrow stem cells in thepresence of a retroviral packaging cell line.
 130. The method of claim53, wherein the population of human bone marrow progenitor cells, insaid culture, expands.
 131. The method of claim 53, wherein said culturemedium contains at least one member selected from the group consistingof human peripheral blood mononuclear cells, human bone marrow cells,human fetal liver cells, and human cord blood cells.
 132. The method ofclaim 131, wherein said human peripheral blood mononuclear cells, humanbone marrow cells, human fetal liver cells, and human cord blood cells,are enriched for said human bone marrow stem cells.
 133. The method ofclaim 53, wherein said cultured human bone marrow stem cells producestable, genetically transformed human bone marrow progenitor cells. 134.The method of claim 53, wherein stable genetically transformed humanleukocytes, platelets, lymphocytes, osteoblasts or osteoclasts areobtained.
 135. The method of claim 53, wherein said cells are culturedin the presence of an immobilized antibody specific to said human bonemarrow stem cells.
 136. The method of claim 53, wherein said human bonemarrow stem cells are found in a human peripheral blood.
 137. The methodof claim 53, wherein said human bone marrow stem cells are found in ahuman fetal liver.
 138. The method of claim 53, wherein said human bonemarrow stem cells are found in a human umbilical cord blood.